Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
DESCRIPTION
GENE SEQUENCE VARIANCES WITH
UTILITY IN DETERMINING THE TREATMENT OF DISEASE
RELATED APPLICATIONS
This application is a CIP of Stanton, U.S. Patent Application Serial No. not
yet
assigned, attorney docket 244/273, filed July 19, 1999, entitled GENE SEQUENCE
VARIANCES WITH UTILITY IN DETERMINING THE TREATMENT OF DISEASE
and claims the benefit of Stanton, U.S. Provisional Application 60/093,484,
filed July 20,
1998, entitled GENE SEQUENCE VARIANCES WITH UTILITY IN DETERMINING
THE TREATMENT OF DISEASE, which are hereby incorporated by reference in their
entireties including drawings and tables.
BACKGROUND OF THE INVENTION
This application concerns the field of mammalian therapeutics and the
selection of
therapeutic regimens utilizing host genetic information, including gene
sequence
variances within the human genome in human populations.
The rate of approval of new drugs that enter human clinical trials is less
than 20%,
despite demonstrated efficacy of said new drugs in preclinical models of human
disease.
In some instances the low response rate in humans is due to genetic
heterogeneity in the
drug target or the pathway mediating the action of the drug. Identification of
the genetic
causes of variable drug response would allow more rational clinical
development of
drugs. Further, many drugs or other treatments approved for use in humans are
known to
have highly variable safety and efficacy in different individuals. A
consequence of such
variability is that a given drug or other treatment may be highly effective in
one
individual, and ineffective or not well tolerated in another individual. Thus,
administration of such a drug to an individual in whom the drug would be
ineffective
would result in wasted cost and time during which the patient's condition may
significantly worsen. Also, administration of a drug to an individual in whom
the drug
would not be tolerated could result in a direct worsening of the patient's
condition and
could even result in the patient's death.
CA 02335649 2001-O1-19
2
WO 00104194 PCT/US99/16440
For some drugs, up to 99% of the measurable variation in selected
pharmacokinetic parameters has been shown to be inherited, or associated with
genetic
factors. Studies have also demonstrated a significant genetic component to
pharmacodynamic variation. For a limited number of drugs, discrete gene
sequence
variances have been identified in specific genes that are involved in drug
action, and these
variances have been shown to account for the variable efficacy or safety of
the drug in
different individuals.
SUMMARY OF THE INVENTION
io
The present invention is concerned generally with the field of treatment of
diseases and conditions in mammals, particularly in humans. It is concerned
with the
genetic basis of inter-patient variation in response to therapy, including
drug therapy.
Specifically, this invention describes the identification of gene sequence
variances useful
15 in the field of therapeutics for optimizing efficacy and safety of drug
therapy for specific
diseases or conditions and for establishing diagnostic tests useful for
improving the
development and use of pharmaceutical products in the clinic. Methods for
identifying
genetic variances and determining their utility in the selection of optimal
therapy for
specific patients are also described, along with probes and related materials
which are
2o useful, for example, in identifying the presence of a particular gene
sequence variance in
cells of an individual. The genes involved in the present invention are those
listed in a
pathway, gene table, list or example herein.
The inventors have determined that the identification of gene sequence
variances
within genes that may be involved in drug action is important for determining
whether
25 genetic variances account for variable drug efficacy and safety and for
determining
whether a given drug or other therapy may be safe and effective in an
individual patient.
Provided in this invention are identifications of genes and sequence variances
which can
be useful in connection with predicting differences in response to treatment
and selection
of appropriate treatment of a disease or condition. Such genes and variances
have utility
3o in pharmacogenetic association studies and diagnostic tests to improve the
use of certain
drugs or other therapies including, but not limited to, the drug classes and
specific drugs
identified in the 1999 Physicians' Desk Reference (53rd edition), Medical
Economics
CA 02335649 2001-O1-19
WO 00/04194 PCTNS99/16440
Data, 1998, or the 1995 United States Pharmacopeia XXIII National Formulary
XVIIZ,
Interpharm Press, 1994, or other sources as described below.
The terms "disease" or "condition" are commonly recognized in the art and
designate the presence of signs and/or symptoms in an individual or patient
that are
generally recognized as abnormal. Diseases or conditions may be diagnosed and
categorized based on pathological changes. Signs may include any objective
evidence of
a disease such as changes that are evident by physical examination of a
patient or the
results of diagnostic tests which may include, among others, laboratory tests
to determine
the presence of variances or variant forms of certain genes in a patient.
Symptoms are
to subjective evidence of disease or a patients condition - i.e, the patients
perception of an
abnormal condition that differs from normal function, sensation, or
appearance, which
may include, without limitations, physical disabilities, morbidity, pain, and
other changes
from the normal condition experienced by an individual. Various diseases or
conditions
include, but are not limited to, those categorized in standard textbooks of
medicine
including, without limitation, textbooks of nutrition, allopathic,
homeopathic, and
osteopathic medicine. In certain aspects of this invention, the disease or
condition is
selected from the group consisting of the types of diseases listed in standard
texts such as
Harrison's Principles of Internal Medicine (14th Ed) by Anthony S. Fauci,
Eugene
Braunwald, Kurt J. Isselbacher, et al. (Editors), McGraw Hill,1997, or Robbins
2o Pathologic Basis of Disease (6th edition) by Ramzi S. Cotran, Vinay Kumar,
Tucker
Collins & Stanley L. Robbins, W B Saunders Co., 1998, or the Diagnostic and
Statistical
Manual of Mental Disorders: Dsm-IV (4th Ed), American Psychiatric Press, 1994
or
other texts described below.
In connection with the methods of this invention, unless otherwise indicated,
the
term "suffering from a disease or condition" means that a person is either
presently
subject to the signs and symptoms, or is more likely to develop such signs and
symptoms
than a normal person in the population. Thus, for example, a person suffering
from a
condition can include a developing fetus, a person subject to a treatment or
environmental
condition which enhances the likelihood of developing the signs or symptoms of
a
3o condition, or a person who is being given or will be given a treatment
which increase the
likelihood of the person developing a particular condition. For example,
tardive
dyskinesia is associated with long-term use of anti-psychotics;
gastrointestinal symptoms,
alopecia and bone marrow suppression are associated with cancer
chemotherapeutic
CA 02335649 2001-O1-19
4
PCT/US99I16440 _
WO 00/04194
regimens, and immunosuppression is associated with agents to limit graft
rejection
following transplantation. Thus, methods of the present invention which relate
to
treatments of patients (e.g., methods for selecting a treatment, selecting a
patient for a
treatment, and methods of treating a disease or condition in a patient) can
include primary
treatments directed to a presently active disease or condition, secondary
treatments which
are intended to cause a biological effect relevant to a primary treatment, and
prophylactic
treatments intended to delay, reduce, or prevent the development of a disease
or
condition, as well as treatments intended to cause the development of a
condition
different from that which would have been likely to develop in the absence of
the
to treatment.
The term "therapy" refers to a process which is intended to produce a
beneficial
change in the condition of a mammal, e.g., a human, often referred to as a
patient. A
beneficial change can, for example, include one or more of restoration of
function,
reduction of symptoms, limitation or retardation of progression of a disease,
disorder, or
condition or prevention, limitation or retardation of deterioration of a
patient's condition,
disease or disorder. Such therapy can involve, for example, nutritional
modifications,
administration of radiation, administration of a drug, behavioral
modifications and
combinations of these, among others.
The term "drug" as used herein refers to a chemical entity or biological
product, or
2o combination of chemical entities or biological products, administered to a
person to treat
or prevent or control a disease or condition. The chemical entity or
biological product is
preferably, but not necessarily a low molecular weight compound, but may also
be a
larger compound, for example, an oligomer of nucleic acids, amino acids, or
carbohydrates including without limitation proteins, oligonucleotides,
ribozymes,
DNAzymes, glycoproteins, lipoproteins, and modifications and combinations
thereof. A
biological product is preferably a monoclonal or polyclonal antibody or
fragment thereof
such as a variable chain fragment cells; or an agent or product arising from
recombinant
technology, such as, without limitation, a recombinant protein, recombinant
vaccine, or
DNA construct developed for therapeutic, e.g., human therapeutic, use. The
term "drug"
3o may include, without limitation, compounds that are approved for sale as
pharmaceutical
products by government regulatory agencies (e.g., U.S. Food and Drug
Administration
(USFDA or FDA), European Medicines Evaluation Agency (EMEA), and a world
regulatory body governing the Intemation Conference of Harmonization (ICH)
rules and
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99116440
guidelines), compounds that do not require approval by government regulatory
agencies,
food additives or supplements including compounds commonly characterized as
vitamins, natural products, and completely or incompletely characterized
mixtures of
chemical entities including natural compounds or purified or partially
purified natural
products. The term "drug" as used herein is synonymous with the terms
"medicine",
"pharmaceutical product", or "product". Most preferably the drug is approved
by a
government agency for treatment of a specific disease or condition.
A "low molecular weight compound" has a molecular weight <5,000 Da, more
preferably <2500 Da, still more preferably <1000 Da, and most preferably <700
Da.
1o Those familiar with drug use in medical practice will recognize that
regulatory
approval for drug use is commonly limited to approved indications, such as to
those
patients afflicted with a disease or condition for which the drug has been
shown to be
likely to produce a beneficial effect in a controlled clinical trial.
Unfortunately, it has
generally not been possible with current knowledge to predict which patients
will have a
beneficial response, with the exception of certain diseases such as bacterial
infections
where suitable laboratory methods have been developed. Likewise, it has
generally not
been possible to determine in advance whether a drug will be safe in a given
patient.
Regulatory approval for the use of most drugs is limited to the treatment of
selected
diseases and conditions. The descriptions of approved drug usage, including
the
2o suggested diagnostic studies or monitoring studies, and the allowable
parameters of such
studies, are commonly described in the "label" or "insert" which is
distributed with the
drug. Such labels or inserts are preferably required by government agencies as
a
condition for marketing the drug and are listed in common references such as
the
Physicians Desk Reference (PDR). These and other limitations or considerations
on the
use of a drug are also found in medical journals, publications such as
pharmacology,
pharmacy or medical textbooks including, without limitation, textbooks of
nutrition,
allopathic, homeopathic, and osteopathic medicine.
Many widely used drugs are effective in a minority of patients receiving the
drug,
particularly when one controls for the placebo effect. For example, the PDR
shows that
3o about 45% of patients receiving Cognex (tacrine hydrochloride) for
Alzheimer's disease
show no change or minimal worsening of their disease, as do about 68% of
controls
(including about 5% of controls who were much worse). About 58% of Alzheimer's
patients receiving Cognex were minimally improved, compared to about 33% of
controls,
CA 02335649 2001-O1-19
6
WO 00/04194 PCTIUS99/16440
while about 2% of patients receiving Cognex were much improved compared to
about
1 % of controls. Thus a tiny fraction of patients had a significant benefit.
Response to
many cancer chemotherapy drugs is even worse. For example, 5-fluorouracil is
standard
therapy for advanced colorectal cancer, but only about 20-40% of patients have
an
objective response to the drug, and, of these, only 1-5% of patients have a
complete
response (complete tumor disappearance; the remaining patients have only
partial tumor
shrinkage). Conversely, up to 20-30% of patients receiving 5-FU suffer serious
gastrointestinal or hematopoietic toxicity, depending on the regimen.
Thus, in a first aspect, the invention provides a method for selecting a
treatrnent
to for a patient suffering from a disease or condition by determining whether
or not a gene
or genes in cells of the patient {in some cases including both normal and
disease cells,
such as cancer cells) contain at least one sequence variance which is
indicative of the
effectiveness of the treatment of the disease or condition. The gene is one
specified
herein, in particular one listed in a Table or list herein. Preferably the at
least one
variance includes a plurality of variances which may provide a haplotype or
haplotypes.
Preferably the joint presence of the plurality of variances is indicative of
the potential
effectiveness of the treatment in a patient having such plurality of
variances. The
plurality of variances may each be indicative of the potential effectiveness
of the
treatment, and the effects of the individual variances may be independent or
additive, or
2o the plurality of variances may be indicative of the potential effectiveness
if at least 2, 3, 4,
or more appear jointly. The plurality of variances may also be combinations of
these
relationships. The plurality of variances may include variances from one, two,
three or
more gene loci.
In a related aspect, the invention concerns a method for providing a
correlation
between a patient genotype and effectiveness of a treatment, by determining
the presence
or absence of a particular known variance or variances in cells of a patient
for a gene of
this invention, and providing a result indicating the expected effectiveness
of a treatment
for a disease or condition. The result may be formulated by comparing the
genotype of
the patient with a list of variances indicative of the effectiveness of a
treatment, e.g.,
3o administration of a drug described herein. The determination may be by
methods as
described herein or other methods known to those skilled in the art.
In some cases, the selection of a method of treatment, i.e., a therapeutic
regimen,
may incorporate selection of one or more from a plurality of medical
therapies. Thus, the
CA 02335649 2001-O1-19
7
PCTNS99o 644
W O 00/04194 -
selection may be the selection of a method or methods which.is/are more
effective or less
effective than certain other therapeutic regimens (with either having varying
safety
parameters). Likewise or in combination with the preceding selection, the
selection may
be the selection of a method or methods which is safer than certain other
methods of
treatment in the patient.
The selection may involve either positive selection or negative selection or
both,
meaning that the selection can involve a choice that a particular method would
be an
appropriate method to use and/or a choice that a particular method would be an
inappropriate method to use. Thus, in certain embodiments, the presence of the
at least
to one variance is indicative that the treatment will be effective or
otherwise beneficial (or
more likely to be beneficial) in the patient. Stating that the treatment will
be effective
means that the probability of beneficial therapeutic effect is greater than in
a person not
having the appropriate presence or absence of particular variances. In other
embodiments, the presence of the at least one variance is indicative that the
treatment will
be ineffective or contra-indicated for the patient. For example, a treatment
may be
contra-indicated if the treatment results, or is more likely to result, in
undesirable side
effects, or an excessive level of undesirable side effects. A determination of
what
constitutes excessive side-effects will vary, for example, depending on the
disease or
condition being treated, the availability of alternatives, the expected or
experienced
2o efficacy of the treatment, and the tolerance of the patient. As for an
effective treatment,
this means that it is more likely that a desired effect will result from the
treatment
administration in a patient with a particular variance or variances than in a
patient who
has a different variance or variances. Also in preferred embodiments, the
presence of the
at least one variance is indicative that the treatment is effective but
results in undesirable
effects or outcomes, e.g., has undesirable side-effects.
In reference to response to a treatment, the term "tolerance" refers to the
ability
of a patient to accept a treatment, based, e.g., on deleterious effects and/or
effects on
lifestyle. Frequently, the term principally concerns the patients perceived
magnitude of
deleterious effects such as nausea, weakness, dizziness, and diarrhea, among
others. Such
3o experienced effects can, for example, be due to general or cell-specific
toxicity, activity
on non-target cells, cross-reactivity on non-target cellular constituents (non-
mechanism
based), and/or side-effects of activity on the target cellular subsitutuent
(mechanism
based), or the cause of toxicity may not be understood. In any of these
circumstances one
CA 02335649 2001-O1-19
WO 00/04194 PCTNS99/16440 -
may identify an association between the undesirable effects and variances in
specific
genes.
Adverse responses to drugs constitute a major medical problem, as shown in two
recent meta-analyses (Lazarou, J. et al, Incidence of adverse drug reactions
in
hospitalized patients: a meta-analysis of prospective studies, JAMA 279:1200-
1205,
1998; Bonn, Adverse drug reactions remain a major cause of death, Lancet
351:1183,
1998). An estimated 2.2 million hospitalized patients in the United Stated had
serious
adverse drug reactions in 1994, with an estimated 106,000 deaths (Lazarou et
al.). To the
extent that some of these adverse events are due to genetically encoded
biochemical
1o diversity among patients in pathways that effect drug action, the
identification of
variances that are predictive of such effects will allow for more effective
and safer drug
use.
In embodiments of this invention, the variance or variant form or forms of a
gene
is/are associated with a specific response to a drug. The frequency of a
specific variance
is or variant form of the gene may correspond to the frequency of an
efficacious response to
administration of a drug. Alternatively, the frequency of a specific variance
or variant
form of the gene may correspond to the frequency of an adverse event resulting
from
administration of a drug. Alternatively the frequency of a specific variance
or variant
form of a gene may not correspond closely with the frequency of a beneficial
or adverse
2o response, yet the variance may still be useful for identifying a patient
subset with high
response or toxicity incidence because the variance may account for only a
fraction of the
patients with high response or toxicity. Preferably, the drug will be
effective in more
than 20% of individuals with one or more specific variances or variant forms
of the gene,
more preferably in 40% and most preferably in >60%. In other embodiments, the
drug
25 will be toxic or create clinically unacceptable side effects in more than
10% of
individuals with one or more variances or variant forms of the gene, more
preferably in
>30%, more preferably in >50%, and most preferably in >70% or in more than
90%.
Also in other embodiments, the method of selecting a treatment includes
eliminating a treatment, where the presence or absence of the at least one
variance is
3o indicative that the treatment will be ineffective or contra-indicated. In
other preferred
embodiments, in cases in which undesirable side-effects may occur or are
expected to
occur from a particular therapeutic treatment, the selection of a method of
treatment can
include identifying both a first and second treatment, where the first
treatment is effective
CA 02335649 2001-O1-19
9
WO 00/04194 PCT/US99/16440
to treat the disease or condition, and the second treatment reduces a
deleterious effect of
the first treatment.
The phrase "eliminating a treatment" refers to removing a possible treatment
from consideration, e.g., for use with a particular patient based on the
presence or absence
of a particular variances) in one or more genes in cells of that patient, or
to stopping the
administration of a treatment which was in the course of administration.
Usually, the treatment will involve the administration of a compound
preferentially active in patients with a form or forms of a gene, where the
gene is one
identified herein. The administration may involve a combination of compounds.
Thus,
to in preferred embodiments, the method involves identifjring such an active
compound or
combination of compounds, where the compound is less active or is less. safe
or both
when administered to a patient having a different form of the gene. In
preferred
embodiments, the compound is a compound in a drug class identified in the 1999
Physicians' Desk Reference (53rd edition), Medical Economics Data, 1998, the
1s PharmaProjects database, the IMS database or identified herein, e.g., in an
exemplary
drug table herein (see, e.g., Examples 6, 8, and 9 and Tables 7 and 9 herein).
Also in preferred embodiments, the method of selecting a treatment involves
selecting a method of administration of a compound, combination of compounds,
or
pharmaceutical composition, for example, selecting a suitable dosage level
and/or
2o frequency of administration, and/or mode of administration of a compound.
The method
of administration can be selected to provide better, preferably maximum
therapeutic
benefit. In this context, "maximum" refers to an approximate local maximum
based on
the parameters being considered, not an absolute maximum.
Also in this context, a "suitable dosage level" refers to a dosage level which
25 provides a therapeutically reasonable balance between pharmacological
effectiveness and
deleterious effects. Often This dosage level is related to the peak or aveage
serum levels
resulting from administration of a drug at the particular dosage level.
Similarly, a "frequency of administration" refers to how often in a specified
time
period a treatment is administered, e.g., once, twice, or three times per day,
every other
3o day, once per week, etc. For a drug or drugs, the frequency of
administration is generally
selected to achieve a pharmacologically effective average or peak serum level
without
excessive deleterious effects (and preferably while still being able to have
reasonable
patient compliance for self administered drugs). Thus, it is desirable to
maintain the
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
serum level of the drug within a therapeutic window of concentrations for the
greatest
percentage of time possible without such deleterious effects as would cause a
prudent
physician to reduce the frequency of administration for a particular dosage
level.
A particular gene or genes can be relevant to more than one disease or
condition,
5 for example, the gene or genes can have a role in the initiation,
development, course,
treatment, treatment outcomes, or health-related quality of life outcomes of a
number of
different diseases, disorders, or conditions. Thus, in preferred embodiments,
the disease
or condition or treatment of the disease or condition is any which involves a
particular
gene. Preferably the gene is a gene identified herein.
to Determining the presence of a particular variance or plurality of variances
in a
particular gene in a patient can be performed in a variety of ways. In
preferred
embodiments, the detection of the presence or absence of at least one variance
involves
amplifying a segment of nucleic acid including at least one of the at least
one variances.
Preferably a segment of nucleic acid to be amplified is 500 nucleotides or
less in length,
more preferably 100 nucleotides or less, and most preferably 45 nucleotides or
less.
Also, preferably the amplified segment or segments includes a plurality of
variances, or a
plurality of segments of a gene or of a plurality of genes.
In another aspect determining the presence of a set of variances in a specific
gene
may entail a haplotyping test that requires allele-specific amplification of a
large DNA
2o segment of no greater than 20,000 nucleotides, preferably no greater than
10,000
nucleotides and more preferably no greater than 5,000 nucleotides.
Alternatively one
allele may be enriched by methods other than amplification prior to
determining
genotypes at specific variant positions on the enriched allele as a way of
determining
haplotypes. Preferably the determination of the presence or absence of a
variance
involves determining the sequence of the variance site or sites by methods
such as chain
terminating DNA sequencing or minisequencing, or by oligonucleotide
hybridization or
by mass spectrometry.
The term "genotype" in the context of this invention refers to the particular
alleleic form of a gene, which can be defined by the particular nucleotides)
present in a
3o nucleic acid sequence at a particular site(s).
In preferred embodiments, the detection of the presence or absence of the at
least
one variance involves contacting a nucleic acid sequence corresponding to one
of the
genes identified above or a product of such a gene with a probe. The probe is
able to
CA 02335649 2001-O1-19
PCT/US99/16440
WO 00/04194 11
distinguish a particular form of the gene or gene product or the presence or a
particular
variance or variances, e.g., by differential binding or hybridization. Thus,
exemplary
probes include nucleic acid hybridization probes, peptide nucleic acid probes,
nucleotide-
containing probes which also contain at least one nucleotide analog, and
antibodies, e.g.,
monoclonal antibodies, and other probes as discussed herein. Those skilled in
the art are
familiar with the preparation of probes with particular specificities. Those
skilled in the
art will recognize that a variety of variables can be adjusted to optimize the
discrimination between two variant forms of a gene, including changes in salt
concentration, temperature, pH and addition of various compounds that affect
the
differential affinity of GC vs. AT base pairs, such as tetramethyl ammonium
chloride.
(See Current Protocols in Molecular Biology by F. M~. Ausubel, R. Brent, R. E.
Kingston,
D. D. Moore, J.G. Seidman, K. Struhl and V. B. Chanda (Editors), John Wiley &
Sons.)
In other preferred embodiments, determining the presence or absence of the at
least one variance involves sequencing at least one nucleic acid sequence. The
t5 sequencing involves sequencing of a portion or portions of a gene and/or
portions of a
plurality of genes which includes at least one variance site, and may include
a plurality of
such sites. Preferably, the portion is 500 nucleotides or less in length, more
preferably
100 nucleotides or less, and most preferably 45 nucleotides or less in length.
Such
sequencing can be carried out by various methods recognized by those skilled
in the art,
including use of dideoxy termination methods (e.g., using dye-labeled dideoxy
nucleotides) and the use of mass spectrometric methods. In addition, mass
spectrometric
methods may be used to determine the nucleotide present at a variance site. In
preferred
embodiments in which a plurality of variances is determined, the plurality of
variances
can constitute a haplotype or haplotypes.
The terms "variant form of a gene", "form of a gene", or "allele" refer to one
specific form of a gene in a population, the specific form differing from
other forms of
the same gene in the sequence of at least one, and frequently more than one,
variant sites
within the sequence of the gene. The sequences at these variant sites that
differ between
different alleles of the gene are termed "gene sequence variances" or
"variances" or
"variants". The term "alternative form" refers to an allele that can be
distinguished from
other alleles by having distinct variances at at least one, and frequently
more than one,
variant sites within the gene sequence. Other terms known in the art to be
equivalent
include mutation and polymorphism, although mutation is often used to refer to
an allele
CA 02335649 2001-O1-19
12
WO 00/04194 PCT/US99/16440
associated with a deleterious phenotype. In preferred aspects of this
invcntion, the
variances are selected from the group consisting of the variances listed in
the variance
tables herein or in a patent or patent application referenced and incorporated
by reference
in this disclosure. In the methods utilizing variance presence or absence,
reference to the
presence of a variance or variances means particular variances, i.e.,
particular nucleotides
at particular polymorphic sites, rather than just the presence of any variance
in the genc.
Variances occur in the human genome at approximately one in every 500 -1,000
bases within the human genome when two alleles are compared. When multiple
alleles
from unrelated individuals are compared the frequency of variant sites
increases. At most
to variant sites there are only two alternative nucleotides involving the
substitution of one
base for another or the insertion/deletion of one or more nucleotides. Within
a gene there
may be several variant sites. Variant forms of the gene or alternative alleles
can be
distinguished by the presence of alternative variances at a single variant
site, or a
combination of several different variances at different sites (haplotypes).
It is estimated that there are 3,300,000,000 bases in the sequence of a single
haploid human genome. All human cells except germ cells are normally diploid.
Each
gene in the genome may span 100-10,000,000 bases of DNA sequence or 100-20,000
bases of mRNA. It is estimated that there are between 60,000 and 120,000 genes
in the
human genome. The "identification" of genetic variances or variant forms of a
gene
2o involves the discovery of variances that are present in a population. The
identification of
variances is required for development of a diagnostic test to determine
whether a patient
has a variant form of a gene that is known to be associated with a disease,
condition, or
predisposition or with the efficacy or safety of the drug. Identification of
previously
undiscovered genetic variances is distinct from the process of "determining"
the status of
known variances by a diagnostic test. The present invention provides exemplary
variances in genes listed in the gene tables, as well as methods for
discovering additional
variances in those genes and a comprehensive written description of such
additional
possible variances. Also described are methods for DNA diagnostic tests to
determine
the DNA sequence at a particular variant site or sites.
3o The process of "identifying" or discovering new variances involves
comparing the
sequence of at least two alleles of a gene, more preferably at least 10
alleles and most
preferably at least 50 alleles, (keeping in mind that each somatic cell has
two alleles).
The analysis of large numbers of individuals to discover variances in the gene
sequence
CA 02335649 2001-O1-19
13
WO 00/04194 PCT/US99/16440
between individuals in a population will result in detection of a greater
fraction of all the
variances in the population. Preferably the process of identifying reveals
whether there is
a variance within the gene; more preferably identifying reveals the location
of the
variance within the gene; more preferably identifying provides knowledge of
the
s sequence of the nucleic acid sequence of the variance, and most preferably
identifying
provides knowledge of the combination of different variances that comprise
specific
variant forms of the gene or alleles. In identifying new variances it is often
useful to
screen different population groups based on racial, ethnic, gender, and/or
geographic
origin because particular variances may differ in frequency between such
groups. It may
to also be useful to screen DNA from individuals with a particular disease or
condition of
interest because they may have a higher frequency of certain variances than
the general
population.
The process of determining involves using diagnostic tests for specific
variances
or variant forms of the gene (or genes) that have been identified within the
gene. It will
15 be apparent that such diagnostic tests can only be performed after
variances and variant
forms of the gene have been identified. Identification of variances can be
performed by a
variety of methods, alone or in combination, including, for example, DNA
sequencing,
SSCP, heteroduplex analysis, denaturing gradient gel electrophoresis (DGGE),
heteroduplex cleavage (either enzymatic as with T4 Endonuclease 7, or chemical
as with
2o osmium tetroxide and hydroxylamine), computational methods (described
herein), and
other methods described herein as well as others known to those skilled in the
art. (See,
for example: Cotton, R.G.H., Slowly but surely towards better scanning for
mutations,
Trends in Genetics 13(2):43-6, 1997, or Current Protocols in Human Genetics by
N. C.
Dracopoli, J. L. Haines, B. R. Korf, D. T. Moir , C. C. Morton, C. E. Seidman,
J.G.
25 Seidman, D. R. Smith and A. Boyle (Editors), John Wiley & Sons.)
In the context of this invention, the term " analyzing a sequence" refers to
determining at least some sequence information about the sequence, e.g.,
determining the
nucleotides present at particular sites in the sequence or determining the
base sequence of
all of a portion of the particular sequence.
3o In the context of this invention, the tenor "haplotype" refers to a cis
arrangement
of two or more polymorphic nucleotides, i.e., variances, on a particular
chromosome, e.g.,
in a particular gene. The haplotype preserves the infornation of the phase of
the
CA 02335649 2001-O1-19
14
WO 00/04194 PCT/US99/16440
polymorphic nucleotides - that is, which set of variances were inherited from
one parent,
and which from the other.
In preferred embodiments of this invention, the frequency of the variance or
variant form of the gene in a population is known. Measures of frequency known
in the
art include "allele frequency", namely the fraction of genes in a population
that have one
specific variance or set of variances. The allele frequencies for any gene
should sum to 1.
Another measure of frequency known in the art is the "heterozygote frequency"
namely,
the fraction of individuals in a population who carry two alleles, or two
forms of a
particular variance or variant form of a gene, one inherited from each parent.
Alternatively, the number of individuals who are homozygous for a particular
form of a
gene may be a useful measure. The relationship between allele frequency,
heterozygote
frequency, and homozygote frequency is described for many genes by the Hardy-
Weinberg equation, which provides the relationship between allele frequency,
heterozygote frequency and homozygote frequency in a freely breeding
population at
I5 equilibrium. Most human variances are substantially in Hardy-Weinberg
equilibrium. In
a preferred aspect of this invention, the allele frequency, heterozygote
frequency, or
homozygote frequency are determined experimentally. Preferably a variance has
an allele
frequency of at least 0.01, more preferably at least 0.05, still more
preferably at least 0.10.
However, the allele may have a frequency as low as 0.001 if the associated
phenotype is a
2o rare form of toxic reaction to the treatment or drug.
In this regard, "population" refers to a geographically, ethnically, racially,
gender,
and/or culturally defined group of individuals or a group of individuals with
a particular
disease or condition or individuals that may be treated with a specific drug.
In most cases
a population will preferably encompass at least ten thousand, one hundred
thousand, one
25 million, ten million, or more individuals, with the larger numbers being
more preferable.
In a preferred aspect of this invention, the population refers to individuals
with a specific
disease or condition that may be treated with a specific drug. In an aspect of
this
invention, the allele frequency, heterozygote frequency, or homozygote
frequency of a
specific variance or variant form of a gene is known. In preferred embodiments
of this
3o invention, the frequency of one or more variances that may predict response
to a
treatment is determined in one or more populations using a diagnostic test.
It should be emphasized that it is currently not generally practical to study
entire
gene sequences in entire populations to establish the association between a
specific
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
disease or condition and a specific variance or variant form of the gene. Such
studies are
commonly performed in controlled clinical trials using a limited number of
patients that
are considered to be representative of the population with the disease.
In the context of this invention, the term "probe" refers to a molecule which
can
s deteciably distinguish between target molecules differing in structure.
Detection can be
accomplished in a variety of different ways depending on the type of probe
used and the
type of target molecule. Thus, for example, detection may be based on
discrimination of
activity levels of the target molecule, but preferably is based on detection
of specific
binding. Examples of such specific binding include antibody binding and
nucleic acid
1o probe hybridization. Thus, for example, probes can include enzyme
substrates,
antibodies and antibody fragments, and nucleic acid hybridization probes.
Thus, in
preferred embodiments, the detection of the presence or absence of the at
least one
variance involves contacting a nucleic acid sequence which includes a variance
site with a
probe, preferably a nucleic acid probe, where the probe preferentially
hybridizes with a
1s form of the nucleic acid sequence containing a complementary base at the
variance site as
compared to hybridization to a form of the nucleic acid sequence having a non-
complementary base at the variance site, where the hybridization is carried
out under
selective hybridization conditions. Such a nucleic acid hybridization probe
may span two
or more variance sites. Unless otherwise specified, a nucleic acid probe can
include one
2o or more nucleic acid analogs, labels or other substituents or moieties so
long as the base-
pairing function is retained.
As is generally understood, administration of a particular treatment, e.g.,
administration of a therapeutic compound or combination of compounds, is
chosen
depending on the disease or condition which is to be treated. Thus, in certain
preferred
zs embodiments, the disease or condition is one for which administration of a
treatment is
expected to provide a therapeutic benefit; in certain embodiments, the
compound is a
compound identified herein, e.g., in a drug table such as Tables 7 and 9.
As used herein, the terms "effective" and "effectiveness" includes both
pharmacological effectiveness and physiological safety. Pharmacological
effectiveness
3o refers to the ability of the treatment to result in a desired biological
effect in the patient.
Physiological safety refers to the level of toxicity, or other adverse
physiological effects
at the cellular, organ and/or organism level (often referred to as side-
effects) resulting
from administration of the treatment. On the other hand, the term
"ineffective" indicates
CA 02335649 2001-O1-19
PCTIUS99/16440
WO 00/04194 16
that a treatment does not provide sufficient pharmacological effect to be
therapeutically
useful, even in the absence of deleterious effects, at least in the total
(unstratified)
population. (Such a treatment may be effective in a subgroup that can be
identified by
the presence of one or more sequence variances or alleles.) "Less effective"
means that
s the treatment results in a therapeutically significant lower level of
pharmacological
effectiveness and/or a therapeutically greater level of adverse physiological
effects.
Thus, in connection with the administration of a drug, a drug which is
"effective
against" a disease or condition indicates that administration in a clinically
appropriate
manner results in a beneficial effect for at least a statistically significant
fraction of
1o patients, such as a improvement of symptoms, a cure, a reduction in disease
load,
reduction in tumor mass or cell numbers, extension of life, improvement in
quality of life,
or other effect generally recognized as positive by medical doctors familiar
with treating
the particular type of disease or condition.
The term "deleterious effects" refers to physical effects in a patient caused
by
15 administration of a treatment which are regarded as medically undesirable.
Thus, for
example, deleterious effects can include a wide spectrum of toxic effects
injurious to
health such as death of normal cells when only death of diseased cells is
desired, nausea,
fever, inability to retain food, dehydration, damage to critical organs such
as renal tubular
necrosis, fatty liver or pulmonary fibrosis, among many others. In this
regard, the term
20 "contra-indicated" means that a treatment results in deleterious effects
such that a
prudent medical doctor treating such a patient would regard the treatment as
unsuitable
for administration. Major factors in such a determination can include, for
example,
availability and relative advantages of alternative treatments, consequences
of non-
treatment, and permanency of deleterious effects of the treatment.
25 It is recognized that many treatment methods, e.g., administration of
certain
compounds or combinations of compounds, produces side-effects or other
deleterious
effects in patients. Such effects can limit or even preclude use of the
treatment method in
particular patients, or may even result in irreversible injury, dysfirnction,
or death of the
patient. Thus, in certain embodiments, the variance information is used to
select both a
3o first method of treatment and a second method of treatment. Usually the
first treatment is
a primary treatment which provides a physiological effect directed against the
disease or
condition or its symptoms. The second method is directed to reducing or
eliminating one
or more deleterious effects of the first treatment, e.g., to reduce a general
toxicity or to
CA 02335649 2001-O1-19
17
WO 00/04194 PCTNS99/16440
reduce a side effect of the primary treatment. Thus, for example, the second
method can
be used to allow use of a greater dose or duration of the first treatment, or
to allow use of
the first treatment in patients for whom the first treatment would not be
tolerated or
would be contra-indicated in the absence of a second method to reduce
deleterious
effects.
In a related aspect, the invention provides a method for selecting a method of
treatment for a patient suffering from a disease or condition by comparing at
least one
variance in at least one gene in the patient, with a list of variances in the
gene or genes
to which are indicative of the effectiveness of at least one method of
treatment. Preferably
the comparison involves a plurality of variances or a haplotype indicative of
the
effectiveness of at least one method of treatment. Also, preferably the list
of variances
includes a plurality of variances.
Similar to the above aspect, in preferred embodiments the at least one method
of
15 treatment involves the administration of a compound effective in at least
some patients
with a disease or condition; the presence or absence of the at least one
variance is
indicative that the treatment will be effective in the patient; and/or the
presence or
absence of the at least one variance is indicative that the treatment will be
ineffective or
contra-indicated in the patient; and/or the treatment is a first treatment and
the presence or
2o absence of the at least one variance is indicative that a second treatment
will be beneficial
to reduce a deleterious effect of the first treatment; and/or the at least one
treatment is a
plurality of methods of treatment. For a plurality of treatments, preferably
the selecting
involves determining whether any of the methods of treatment will be more
effective than
at least one other of the plurality of methods of treatment. Yet other
embodiments are
25 provided as described for the preceding aspect in connection with methods
of treatment
using administration of a compound; treatment of various diseases, and
variances in
particular genes.
In the context of variance information in the methods of this invention, the
term
"list" refers to one or more variances which have been identified for a series
or genes of
3o potential importance in accounting for inter-individual variation in
treatment response.
Preferably there is a plurality of variances for the gene or genes, preferably
a plurality of
variances for a particular gene. Preferably the list is recorded in written or
electronic
form. For example, variances are recorded in Tables 3, 4, and 10 and
additional gene
CA 02335649 2001-O1-19
I8
WO 00/04194 PCT/US99/16440
variance identification tables herein in a form which allows comparison with
other
variance information.
In addition to the basic method of treatment, often the mode of administration
of a
given compound as a treatment for a disease or condition in a patient is
significant in
determining the course and/or outcome of the treatment for the patient. Thus,
the
invention also provides a method for selecting a method of administration of a
compound
to a patient suffering from a disease or condition, by determining the
presence or absence
of at least one variance in cells of the patient in a gene which is a gene
selected from the
1o genes identified in a gene table or list below, where such presence or
absence is indicative
of an appropriate method of administration of the compound: Preferably, the
selection of
a method of treatment (a treatment regimen) involves selecting a dosage level
or
frequency of administration or route of administration of the compound or
combinations
of those parameters. In preferred embodiments, two or more compounds are to be
15 administered, and the selecting involves selecting a method of
administration for one,
two, or more than two of the compounds, jointly, concurrently, or separately.
As
understood by those skilled in the art, such plurality of compounds is often
used in
combination therapy, and thus may be formulated in a single drug, or may be
separate
drugs administered concurrently, serially, or separately. Other embodiments
are as
2o indicated above for selection of second treatment methods, methods of
identifying
variances, and methods of treatment as described for aspects above.
In another aspect, the invention provides a method for selecting a patient for
administration of a method of treatment for a disease or condition, or of
selecting a
25 patient for a method of administration of a treatment, by comparing the
presence or
absence of at least one variance in a gene as identified above in cells of a
patient, with a
list of variances in the gene, where the presence or absence of the at least
one variance is
indicative that the treatment or method of administration will be effective in
the patient.
If the at least one variance is present in the patient's cells, then the
patient is selected for
3o administration of the treatment.
In preferred embodiments, the disease or the method of treatment is as
described in
aspects above, specifically including, for example, those described for
selecting a method
of treatment
CA 02335649 2001-O1-19
19
WO 00/04194 PCT/US99/16440
In another aspect, the invention provides a method for identifying a subset of
patients with enhanced or diminished response or tolerance to a treatment
method or a
method of administration of a treatment where the treatment is for a disease
or condition
in the patient. The method involves correlating one or more variances in one
or more
genes in a plurality of patients with response to a treatment or a method of
administration
of a treatment. The correlation may be performed by determining the one or
more
variances in the one or more genes in the plurality of patients and
correlating the presence
or absence of each of the variances (alone or in various combinations) with
the patient's
response to treatment. The variances may be previously laiown to exist or may
also be
l0 determined in the present method or combinations of prior information and
newly
determined information may be used. The enhanced or diminished response should
be
statistically significant, preferably such that p = 0.10 or less, more
preferably 0.05 or less,
and most preferably 0.02 or less. A positive correlation between the presence
of one or
more variances and an enhanced response to treatment is indicative that the
treatment is
15 particularly effective in the group of patients having those variances. A
positive
correlation of the presence of the one or more variances with a diminished
response to the
treatment is indicative that the treatment will be less effective in the group
of patients
having those variances. Such information is useful, for example, for selecting
or de-
selecting patients for a particular treatment or method of administration of a
treatment, or
2o for demonstrating that a gmup of patients exists for which the treatment or
method of
treatment would be particularly beneficial or contra-indicated. Such
demonstration can
be beneficial, for example, for obtaining government regulatory approval for a
new drug
or a new use of a drug.
In preferred embodiments, the variances are in particular genes, or are
particular
25 variances described herein. Also, preferred embodiments include drugs,
treatments,
variance identification or determination, determination of effectiveness,
lists, and/or
diseases as described for aspects above or otherwise described herein.
In preferred embodiments, the correlation of patient responses to therapy
according to patient genotype is carried out in a clinical trial, e.g., as
described herein
30 according to any of the variations described.. Detailed description of
methods for
associating variances with clinical outcomes using clinical trials are
provided below.
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
As indicated above, in aspects of this invention involving selection of a
patient for
a treatment, selection of a method or mode of administration of a treatment,
and selection
of a patient for a treatment or a method of treatment, the selection may be
positive
selection or negative selection. Thus, the methods can include eliminating a
treatment for
a patient, eliminating a method or mode of administration of a treatment to a
patient, or
elimination of a patient for a treatment or method of treatment.
Also, in methods involving identification and/or comparison of variances
present
in a gene of a patient, the methods can involve such identification or
comparison for a
plurality of genes. Preferably, the genes are fimctionally related to the same
disease or
l0 condition, or to the aspect of disease pathophysiology that is being
subjected to
pharmacological manipulation by the treatment (e.g. a drug), or to the
activation or
inactivation of the drug, and more preferably the genes are involved in the
same
biochemical process or pathway.
15 In another aspect, the invention provides a method for identifying the
forms of a
gene in an individual, where the gene is one specified as for aspects above,
by
determining the presence or absence of at least one variance in the gene. In
preferred
embodiments, the at least one variance includes at least one variance selected
fibm the
group of variances identified in variance tables herein. Preferably, the
presence or
2o absence of the at least one variance is indicative of the effectiveness of
a therapeutic
treatment in a patient suffering from a disease or condition and having cells
containing
the at least one variance.
The presence or absence of the variances can be determined in any of a variety
of
ways as recognized by those skilled in the art. For example, the nucleotide
sequence of at
least one nucleic acid sequence which includes at least one variance site (or
a
complementary sequence) can be determined, such as by chain termination
methods,
hybridization methods or by mass spectrometric methods. Likewise, in preferred
embodiments, the determining involves contacting a nucleic acid sequence or a
gene
product of one of one of the genes with a probe which specifically identifies
the presence
3o or absence of a form of the gene. For example, a pmbe, e.g., a nucleic acid
probe, can be
used which specifically binds, e.g., hybridizes, to a nucleic acid sequence
corresponding
to a portion of the gene and which includes at least one variance site under
selective
CA 02335649 2001-O1-19
PCT/US99/16440
WO 00/04194 21
binding conditions. As described for other aspects, determining the presence
or absence
of at least two variances can constitute determining a haplotype or
haplotypes.
Other preferred embodiments involve variances related to types of treatment,
drug
responses, diseases, nucleic acid sequences, and other items related to
variances and
variance determination as described for aspects above.
In yet another aspect, the invention provides a pharmaceutical composition
which
includes a compound which has a differential effect in patients having at
least one copy,
or alternatively, two copies of a form of a gene as identified for aspects
above and a
to pharmaceutically acceptable carrier, excipient, or diluent. The composition
is adapted to
be preferentially effective to treat a patient with cells containing the one,
two, or mare
copies of the form of the gene.
In preferred embodiments of aspects involving pharmaceutical compositions,
active compounds, or drugs, the material is subject to a regulatory limitation
or restriction
15 on approved uses or indications, e.g., by the U.S. Food and Drug
Administration (FDA),
limiting approved use of the composition to patients having at least one copy
of the
particular form of the gene which contains at least one variance.
Alternatively, the
composition is subject to a regulatory limitation or restriction on approved
uses indicating
that the composition is not approved for use or should not be used in patients
having at
20 least one copy of a form of the gene including at least one variance. Also
in preferred
embodiments, the composition is packaged, and the packaging includes a label
or insert
indicating or suggesting beneficial therapeutic approved use of the
composition in
patients having one or two copies of a form of the gene including at least one
variance.
Alternatively, the label or insert limits approved use of the composition to
patients having
25 zero or one or two copies of a form of the gene including at least one
variance. The latter
embodiment would be likely where the presence of the at least one variance in
one or two
copies in cells of a patient means that the composition would be ineffective
or deleterious
to the patient. Also in preferred embodiments, the composition is indicated
for use in
treatment of a disease or condition which is one of those identified for
aspects above.
3o Also in preferred embodiments, the at least one variance includes at least
one variance
from those identified herein.
The term "packaged" means that the drug, compound, or composition is prepared
in a manner suitable for distribution or shipping with a box, vial, pouch,
bubble pack, or
CA 02335649 2001-O1-19
22
WO 00/04194 PCT/US99116440
other protective container, which may also be used in combination. The
packaging may
have printing on it and/or printed material may be included in the packaging.
In preferred embodiments, the drug is selected from the drug classes or
specific
exemplary drugs identified in an example, in a table or list herein, and is
subject to a
regulatory limitation or suggestion or warning as described above that limits
or suggests
limiting approved use to patients having specific variances or variant forms
of a gene .
identified in Examples or in a gene list provided below in order to achieve
maximal
benefit and avoid toxicity or other deleterious effect.
A pharmaceutical composition can be adapted to be preferentially effective in
a
1o variety of ways. In some cases, an active compound is selected which was
not previously
'known to be differentially active, or which was not previously recognized as
a potential
therapeutic compound. In some cases, the concentration of an active compound
which
has differential activity can be adjusted such that the composition is
appropriate for
administration to a patient with the specified variances. For example, the
presence of a
15 specified variance may allow or require the administration of a much larger
dose, which
would not be practical with a previously utilized composition. Conversely, a
patient may
require a much lower dose, such that administration of such a dose with a
prior
composition would be impractical or inaccurate. Thus, the composition may be
prepared
in a higher or lower unit dose form, or prepared in a higher or lower
concentration of the
zo active compound or compounds. In yet other cases, the composition can
include
additional compounds needed to enable administration of a particular active
compound in
a patient with the specified variances, which was not in previous
compositions, e.g.,
because the majority of patients did not require or benefit from the added
component.
The term "differential" or "differentially" generally refers to a
statistically
25 significant different level in the specified property or effect.
Perferably, the difference is
also functionally significant. Thus, "differential binding or hybridization"
is sufficient
difference in binding or hybridization to allow discrimination using an
appropriate
detection technique. Likewise, "differential effect" or "differentially
active" in
connection with a therapeutic treatment or drug refers to a difference in the
level of the
3o effect or activity which is distinguishable using relevant parameters and
techniques for
the effect or activity being considered. Preferably the difference in effect
or activity is
also sufficient to be clinically significant, such that a corresponding
difference in the
CA 02335649 2001-O1-19
23
WO 00/04194 PCTlUS99/16440
course of treatment or treatment outcome would be expected, at least on a
probabilistic
basis.
Also usefully provided in the present invention are probes which specifically
recognize a nucleic acid sequence corresponding to a variance or variances in
a gene or a
product expressed from the gene, and are able to distinguish a variant form of
the
sequence or gene or gene product from one or more other variant forms of that
sequence,
gene, or gene product under selective conditions. Those skilled in the art
recognize and
understand the identification or determination of selective conditions for
particular probes
to or types of probes. An exemplary type of probe is a nucleic acid
hybridization probe,
which will selectively bind under selective binding conditions to a nucleic
acid sequence
or a gene product corresponding to one or the genes identified for aspects
above. Another
type of probe is a peptide or protein, e.g., an antibody or antibody fragment
which
specifically or preferentially binds to a polypeptide expressed from a
particular form of a
15 gene as characterized by the presence or absence of at least one variance.
Thus, in
another aspect, the invention concerns such probes. In the context of this
invention, a
"probe" is a molecule, commonly a nucleic acid, though also potentially a
protein,
carbohydrate, polymer, or small molecule, that is capable of binding to one
variance or
variant form of the gene or gene product to a greater extent than to a form of
the gene
20 having a different base at one or more variance sites, such that the
presence of the
variance or variant form of the gene can be determined. Preferably the probe
distinguishes at least one variance identified in Examples, tables or lists
below.
Preferably the probe also has specificity for the particular gene or gene
product, at least to
an extent such that binding to other genes or gene products does not prevent
use of the
25 assay to identify the presence or absence of the particular variance or
variances of
interest.
In preferred embodiments,the probe is an antibody or antibody fragment. Such
antibodies may be polyclonal or monoclonal antibodies, and can be prepared by
methods
well-known in the art. In preferred embodiments, the probe is a nucleic acid
probe at
30 least 15, preferably at least 17 nucleotides in length, more preferably at
least 20 or 22 or
25, preferably 500 or fewer nucleotides in length, more preferably 200 or 100
or fewer,
still more preferably 50 or fewer, and most preferably 30 or fewer. In
preferred
embodiments, the probe has a length in a range between from any one of the
above
CA 02335649 2001-O1-19
24
WO 00/04194 PCT/US99/16440
lengths to any other of the above lengths (including endpoints). .The probe
specifically
hybridizes under selective hybridization conditions to a nucleic acid sequence
corresponding to a portion of one of the genes identified in connection with
above
aspects. The nucleic acid sequence includes at least one and preferably two or
morc
s variance sites. Also in preferred embodiments, the probe has a detectable
label,
preferably a fluorescent label. A variety of other detectable labels are known
to those
skilled in the art. Such a nucleic acid probe can also include one or more
nucleic acid
analogs.
In preferred embodiments, the probe is an antibody or antibody fragment which
to specifically binds to a gene product expressed from a form of one of the
above genes,
where the foan~of the gene has at least one specific variance with a
particular base at the
variance site, and preferably a plurality of such variances.
In connection with nucleic acid probe hybridization, the term "specifically
hybridizes" indicates that the probe hybridizes to a sufficiently greater
degree to the
~ s target sequence than to a sequence having a mismatched base at at least
one variance site
to allow distinguishing such hybridization. The term "specifically hybridizes"
thus
means that the probe hybridizes to the target sequence, and not to non-target
sequences, at
a level which allows ready identification of probe/target sequence
hybridization under
selective hybridization conditions. Thus, "selective hybridization conditions"
refer to
2o conditions which allow such differential binding. Similarly, the terms
"specifically
binds" and "selective binding conditions" refer to such differential binding
of any type
of probe, e.g., antibody probes, and to the conditions which allow such
differential
binding. Typically hybridization reactions to determine the status of variant
sites in
patient samples are carried out with two different probes, one specific for
each of the
25 (usually two) possible variant nucleotides. The complementary information
derived from
the two separate hybridization reactions is useful in corroborating the
results.
Likewise, the invention provides an isolated, purified or enriched nucleic
acid
sequence of 15 to 500 nucleotides in length, preferably 15 to 100 nucleotides
in length,
30 more preferably 15 to 50 nucleotides in length, and most preferably 15 to
30 nucleotides
in length, which has a sequence which corresponds to a portion of one of the
genes
identified for aspects above. Preferably the lower limit for the preceding
ranges is 17, 20,
22, or 25 nucleotides in length. In other embodiments, the nucleic acid
sequence is 30 to
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/1b440
300 nucleotides in length, or 45 to 200 nucleotides in length, or 45 to 100
nucleotides in
length. The nucleic acid sequence includes at least one variance site. Such
sequences can,
for example, be amplification products of a sequence which spans or includes a
variance
site in a gene identified herein. Likewise, such a sequence can be a primer
which is able
to bind to or extend through a variance site in such a gene. Yet another
example is a
nucleic acid hybridization probe comprised of such a sequence. In such probes,
primers,
and amplification products, the nucleotide sequence can contain a sequence or
site
corresponding to a variance site or sites, for example,-a variance site
identified herein.
Preferably the presence or absence of a particular variant form in the
heterozygous or
1o homozygous state is indicative of the effectiveness of a method of
treatment in a patient.
Typically primers are utilized in pairs. Primers can be designed oar selected
by
methods well=known to those skilled in the art based on nucleotide sequences
corresponding to at least a portion or a gene identified herein. The primer or
primers
hybridizes to or allows amplification (e.g., using the polymerise chain
reaction) through a
15 nucleic acid sequence containing at least one sequence variance. Preferably
such primers
hybridize to a sequence not more than 300 nucleotides, more preferably not
more than
200 nucleotides, still more preferably not more than 100 nucleotides, and most
preferably
not more than 50 nucleotides away from a variance site which is to be
analyzed.
Preferably, a primer is 100 nucleotides or fewer in length, more preferably 50
nucleotides
20 or fewer, still more preferable 30 nucleotides or fewer, and most
preferably 20 or fewer
nucleotides in length.
In reference to nucleic acid sequences which "correspond" to a gene, the term
"correspond" refers to a nucleotide sequence relationship, such that the
nucleotide
sequence has a nucleotide sequence which is the same as the reference gene or
an
25 indicated portion thereof, or has a nucleotide sequence which is exactly
complementary
in normal Watson-Crick base pairing, or is an RNA equivalent of such a
sequence, e.g., a
mRNA, or is a cDNA derived from an mRNA of the gene.
In a related aspect, the invention provides a kit containing at least one
probe or at
least one primer or both (e.g., as described above) corresponding to a gene or
genes of
this invention. The kit is preferably adapted and configured to be suitable
for
identification of the presence or absence of a particular variance or
variances, which can
include or consist of sequence a nucleic acid sequence corresponding to a
portion of a
CA 02335649 2001-O1-19
26
WO 00/04194 PCT/US99/16440
gene. The kit may also contain a plurality of either or both of such probes
and/or primers,
e.g., 2, 3, 4, 5, 6, or more of such probes and/or primers. Preferably the
plurality of
probes and/or primers are adapted to provide detection of a plurality of
different sequence
variances in a gene or plurality of genes, e.g., in 2, 3, 4, 5, or more genes
or to sequence a
nucleic acid sequence including at least one variance site in a gene or genes.
Preferably
one or more of the variance or variances to be detected are correlated with
variability in a
treatment response or tolerance, and are preferably indicative of an effective
response to a
treatment. In preferred embodiments, the kit contains components (e.g., probes
and/or
primers) adapted or useful for detection of a plurality of variances (which
may be in one
1o or more genes) indicative of the effectiveness of at least one treatment,
preferably of a
plurality of different treatments for a particular disease or condition. It
may also be
desirable to provide a kit containing components adapted or useful to allow
detection of a
plurality of variances indicative of the effectiveness of a treatment or
treatment against a
plurality of diseases. The kit may also optionally contain other components,
preferably
other components adapted for identifying the presence of a particular variance
or
variances. Such additional components can, for example, independently include
a buffer
or buffers, e.g., amplification buffers and hybridization buffers, which may
be in liquid or
dry form, a DNA polymerise, e.g., a polymerise suitable for carrying out PCR,
and
deoxy nucleotide triphosphases (dNTPs). Preferably a probe includes a
detectable label,
2o e.g., a fluorescent label, enzyme label, light scattering label, or other
label. Preferably the
kit includes a nucleic acid or polypeptide array. The array may, for example,
include a
plurality of different antibodies, a plurality of different nucleic acid
sequences. Sites in
the array can allow capture and/or detection of nucleic acid sequences or gene
products
corresponding to different variances in one or more different genes.
Preferably the array
is arranged to provide variance detection for a plurality of variances in one
or more genes
which correlate with the effectiveness of one or more treatments of one or
more diseases.
The kit may also optionally contain instructions for use, which can include a
listing of the variances correlating with a particular treatment or treatments
for a disease
of diseases.
Preferably the kit components are selected to allow detection of a variance
described herein, and/or detection of a variance indicative of a
treatment,e.g.,
administration of a drug, pointed out herein.
CA 02335649 2001-O1-19
27
WO 00/04194 PCT/US99/16440
Additional configurations for kits of this invention will be apparent to those
skilled in the art.
In another aspect, the invention provides a method for determining a genotype
of
an individual in relation to one or more variances in one or more of the genes
identified in
above aspects by using mass spectrometric determination of a nucleic acid
sequence
which is a portion of a gene identified for other aspects of this invention or
a
complementary sequence. Such mass spectrometric methods are known to those
skilled
in the art. In preferred embodiments, the method involves determining the
presence or
to absence of a variance in a gene; determining the nucleotide sequence of the
nucleic acid
sequence; the nucleotide sequence is 100 nucleotides or less in length,
preferably 50 or
less, more preferably 30 or less, and still more preferably 20 nucleotides or
less. In
general, such a nucleotide sequence includes at least one variance site,
preferably a
variance site which is informative with respect to the expected response of a
patient to a
t 5 treatment as described for above aspects.
As indicated above, many therapeutic compounds or combinations of compounds
or pharmaceutical compositions show variable efficacy and/or safety in various
patients
in whom the compound or compounds is administered. Thus, it is beneficial to
identify
2o variances in relevant genes, e.g., genes related to the action or toxicity
of the compound
or compounds. Thus, in a fiuther aspect, the invention provides a method for
determining
whether a compound has a differential effect due to the presence or absence of
at least
one variance in a gene or a variant form of a gene, where the gene is a gene
identified for
aspects above.
25 The method involves identifying a first patient or set of patients
suffering from a
disease or condition whose response to a treatment differs from the response
(to the same
treatment) of a second patient or set of patients suffering from the same
disease or
condition, and then determining whether the frequency of at least one variance
in at least
one gene differs in frequency between the first patient or set of patients and
the second
3o patient or set of patients. A correlation between the presence or absence
of the variance
or variances and the response of the patient or patients to the treatment
indicates that the
variance provides information about variable patient response. In general, the
method
will involve identifying at least one variance in at least one gene. An
alternative
CA 02335649 2001-O1-19
28
WO 00/04194 PCT/US99/16440
approach is to identify a first patient or set of patients suffering from a
disease or
condition and having a particular genotype, haplotype or combination of
genotypes or
haplotypes, and a second patient or set of patients suffering from the same
disease or
condition that have a genotype or haplotype or sets of genotypes or haplotypes
that differ
in a specific way from those of the first set of patients. Subsequently the
extent and
magnitude of clinical response can be compared between the first patient or
set of patients
and the second patient or set of patients. A correlation between the presence
or absence
of a variance or variances or haplotypes and the response of the patient or
patients to the
treatment indicates that the variance provides information about variable
patient response
1o and is useful for the present invention.
The method can utilize a variety of different informative comparisons to
identify
correlations. For example a plurality of pairwise comparisons of treatment
response and
the presence or absence of at least one variance can be performed for a
plurality of
patients. Likewise, the method can involve comparing the response of at least
one patient
homozygous for at least one variance with at least one patient homozygous for
the
alternative form of that variance or variances. The method can also involve
comparing
the response of at least one patient heterozygous for at least one variance
with the
response of at least one patient homozygous for the at least one variance.
Preferably the
heterozygous patient response is compared to both alternative homozygous
forms, or the
2o response of heterozygous patients is grouped with the response of one class
of
homozygous patients and said group is compared to the response of the
alternative
homozygous group.
Such methods can utilize either retrospective or prospective information
concerning treatment response variability. Thus, in a preferred embodiment, it
is
previously known that patient response to the method of treatment is variable.
Also in preferred embodiments, the disease or condition is as for other
aspects of
this invention; for example, the treatment involves administration of a
compound or
pharmaceutical composition.
In preferred embodiments, the method involves a clinical trial, e.g., as
described
herein. Such a trial can be arranged, for example, in any of the ways
described herein,
e.g., in the Detailed Description.
CA 02335649 2001-O1-19
29
WO 00/04194 PCT/US99/I6440
The present invention also provides methods of treatment of a disease or
condition. Such methods combine identification of the presence or absence of
particular
variances with the administration of a compound; identification of the
presence of
particular variances with selection of a method of treatment and
administration of the
treatment; and identification of the presence or absence of particular
variances with
elimination of a method of treatment based on the variance information
indicating that the
treatment is likely to be ineffective or contra-indicated, and thus selecting
and
administering an alternative treatment effective against the disease or
condition. Thus,
preferred embodiments of these methods incorporate preferred embodiments of
such
methods as described for such sub-aspects.
As used herein, a "gene" is a sequence of DNA present in a cell that directs
the
expression of a "biologically active" molecule or "gene product", most
commonly by
transcription to produce RNA and translation to produce protein. The "gene
product' is
most commonly a RNA molecule or protein or a RNA or protein that is
subsequently
modified by reacting with, or combining with, other constituents of the cell.
Such
modifications may include, without limitation, modification of proteins to
form
glycoproteins, lipoproteins, and phosphoproteins, or other modifications known
in the art.
RNA may be modified without limitation by complexing with proteins,
polyadenylation,
splicing, capping or export from the nucleus. The term "gene product" refers
to any
product directly resulting from transcription of a gene. In particular this
includes partial,
precursor, and mature transcription products (i.e, pre-mRNA and mRNA), and
translation
products with or without further processing including, without limitation,
lipidation,
phosphorylation, glycosylation, or combinations of such processing
The term "gene involved in the origin or pathogenesis of a disease or
condition"
refers to a gene that harbors mutations that contribute to the cause of
disease, or
variances that affect the progression of the disease or expression of specific
characteristic
of the disease. The term also applies to genes involved in the synthesis,
accumulation, or
elimination of products that are involved in the origin or pathogenesis of a
disease or
condition including, without limitation, proteins, lipids, carbohydrates,
hormones, or
3o small molecules.
The term "gene involved in the action of a drug" refers to any gene whose gene
product affects the efficacy or safety of the drug or affects the disease
process being
treated by the drug, and includes, without limitation, genes that encode gene
products that
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/Ib440
are targets for drug action, gene products that are involved in the
metabolism, activation
or degradation of the drug, gene products that are involved in the
bioavailability or
elimination of the drug to the target, gene products that affect biological
pathways that, in
turn, affect the action of the drug such as the synthesis or degradation of
competitive
substrates or allosteric effectors or rate limiting reaction, or,
alternatively, gene products
that affect the pathophysiology of the disease process. (Particular variances
in the latter
category of genes may be associated with patient groups in whom disease
etiology is
more or less susceptible to amelioration by the drug. For example, there are
several
pathophysiological mechanisms in hypertension, and depending on the dominant
l0 mechanism in a given patient, that patient may be more or less likely than
the average
hypertensive patient to respond to a drug that primarily. targets one
pathophysiological
mechanism. The relative importance of different pathophysiological mechanisms
in
individual patients is likely to be affected by variances in genes associated
with the
disease pathophysiology. The "action" of a drug refers to its effect on
biological products
15 within the body. The action of a drug also refers to its effects on the
signs or symptoms
of a disease or condition, or effects of the drug that are unrelated to the
disease or
condition leading to unanticipated effects on other processes. Such
unanticipated
processes often lead to adverse events or toxic effects. The terms "adverse
event" or
"toxic" event" are known in the art and include, without limitation, those
listed in the
2o FDA reference system for adverse events.
In accordance with the aspects above and the Detailed Description below, there
is
also described for this invention an approach or method for developing drugs
that are
explicitly indicated for, and/or for which approved use is restricted to
individuals in the
population with specific variances or combinations of variances, as determined
by
25 diagnostic tests for variances or variant forms of certain genes involved
in the disease or
condition or involved in the action of the drug. Such drugs may provide more
effective
treatment for a disease or condition in a population identified or
characterized with the
use of a diagnostic test for a specific variance or variant form of the gene
if the gene is
involved in the action of the drug or in determining a characteristic of the
disease or
30 condition. Such drugs may be developed using the diagnostic tests for
specific variances
or variant forms of a gene to determine the inclusion of patients in a
clinical trial.
Thus, the invention also provides a method for producing a pharmaceutical
composition by identifying a compound which has differential activity against
a disease
CA 02335649 2001-O1-19
WO 00/04194 31 PCTNS99/16440
or condition in patients having at least one variance in a gene,,compounding
the
pharmaceutical composition by combining the compound with a pharmaceutically
acceptable carrier, excipient, or diluent such that the composition is
preferentially
effective in patients who have at least one copy of the variance or variances.
In some
s cases, the patient has two copies of the variance or variances. In preferred
embodiments,
the disease or condition, gene or genes, variances, methods of administration,
or method
of determining the presence or absence of variances is as described for other
aspects of
this invention.
Similarly, the invention provides a method forproducing a pharmaceutical agent
~o by identifying a compound which has differential activity against a disease
or condition
in patients having at least one copy of a form of a gene having at least one
variance and
synthesizing the compound in an amount sufficient to provide a pharmaceutical
effect in
a patient suffering from the disease or condition. The compound can be
identified by
conventional screening methods and its activity confirmed. For example,
compound
15 libraries can be screened to identify compounds which differentially bind
to products of
variant fonms of a particular gene product, or which differentially affect
expression of
variant forms of the particular gene, or which differentially affect the
activity of a product
expressed from such gene. Preferred embodiments are as for the preceding
aspect.
In another aspect, the invention provides a method of treating a disease or
2o condition in a patient by selecting a patient whose cells have an allele of
a gene selected
from the genes listed herein, preferably in Tables 2, 6, or 8. The allele
contains at least
one variance correlated with more effective response to a treatment of the
disease or
condition, or tolerance of a treatment, e.g., a treatment with a drug or a
drug of a class
indicated herein.
25 Preferably the allele contains a variance as shown in Tables 2, 6, or 8 or
other
variance table herein. Also preferably, the altering involves administering to
the patient a
compound preferentially active on at least one but less than all alleles of
the gene.
Preferred embodiments include those as described above for other aspects of
treating a
disease or condition.
3o In a further aspect, the invention provides a method for determining a
method of
treatment effective to treat a disease or condition by altering the level of
activity of a
product of an allele of a gene selected from the genes listed in Table 2, 6,
or 8, and
determining whether that alteration provides a differential effect related to
reducing or
CA 02335649 2001-O1-19
WO 00/04194 32 PCT/US99/1Cr440
alleviating a disease or condition as compared to at least one alternative
allele or an
alteration in toxicity or tolerance of the treatment by a patient or patients.
The presence
of such a differential effect indicates that altering that Level of activity
provides at least
part of an effective treatment for the disease or condition.
Preferably the determining is carried out in a clinical trial, e.g., as
described above
and/or in the Detailed Description below.
In still another aspect, the invention provides a method for evaluating
differential
efficacy of or tolerance to a treatment in a subset of patients who have a
particular
variance or variances in at least one gene by utilizing a clinical trial. In
preferred
to embodiments, the clinical trial is a Phase I, II, III, or IV trial.
Preferred embodiments
include the stratifications and/or analyses as described below in the Detailed
Description.
In yet another aspect, the invention provides a method for identifying at
least one
variance in at least one gene using computer-based sequence analysis or
variance
scanning as known to those skilled in the art.
Preferably the at least one gene is a plurality of genes, preferably at least
10, 20,
50, 100, 200, 500, 1000, 5000, 10,000, or even more. Preferably sequence
andlor
variance information on the plurality of genes is acumulated in one database
or a set of
commonly accessible databases within a single local computer network or on a
single
computer.
In yet another aspect, the invention provides experimental methods for finding
additional variances in any of the genes provided in the table of Table 2, 6,
or 8. In
addition to the sequence analysis method, a number of experimental methods can
also
beneficially be used to identify variances. Thus the invention provides
methods for
producing cDNA (e.g., example 13) or genomic DNA and detecting additional
variances
in the genes provided in Table 2, b, or 8 using the single strand conformation
polymorphism (SSCP) method (Example 14), the T4 Endonuclease VII method
(Example
15) or DNA sequencing (Example 16) or other methods pointed out below. The
application of these methods to the identified genes will provide
identification of
additional variances that can affect inter-individual variation in drug or
other treatment
response. One skilled in the art will recognize that many methods for
experimental
variance detection have been described (in addition to the exemplary methods
of
examples 14, 15 and 16) which can be utilized. These additional methods
include
CA 02335649 2001-O1-19
WO 00/04194 33 PCT/US99/16440
chemical cleavage of mismatches (see, e.g., Ellis TP, et al., Chemical
cleavage of
mismatch: a new look at an established method. Human Mutation 11(5):345-53,
1998),
denaturing gradient gel electrophoresis (see, e.g., Van Orsouw NJ, et al.,
Design and
application of 2-D DGGE-based gene mutational scanning tests. Genet Anal. 14(5-
6):205-13, 1999) and heteroduplex analysis (see, e.g., Ganguly A, et al.,
Conformation-
sensitive gel electrophoresis for rapid detection of single-base differences
in double-
stranded PCR products and DNA fragments: evidence for solvent-induced bends in
DNA
heteroduplexes. Proc Natl Acad Sci USA. 90 (21 ):10325-9, 1993).
to In embodiments any of the above methods involving determination of the
presence or absence of a particular variance or variances, the method
preferably involves
determining the presence or absence using a cell sample from an individual or
individuals. Thus, the methods can also involve obtaining a cell sample from
an
individual. The cell sample can be any of a variety of different cells, e.g.,
blood cells skin
15 cells, muscle cells, normal cells, or cancer cells.
By "comprising" is meant including, but not limited to, whatever follows the
word
"comprising". Thus, use of the teen "comprising" indicates that the listed
elements are
required or mandatory, but that other elements are optional and may or may not
be
2o present. By "consisting of is meant including, and limited to, whatever
follows the
phrase "consisting of'. Thus, the phrase "consisting of indicates that the
listed elements
are required or mandatory, and that no other elements may be present. By
"consisting
essentially of is meant including any elements listed after the phrase, and
limited to
other elements that do not interfere with or contribute to the activity or
action specified in
25 the disclosure for the listed elements. Thus, the phrase "consisting
essentially of
indicates that the listed elements are required or mandatory, but that other
elements are
optional and may or may not be present depending upon whether or not they
affect the
activity or action of the listed elements.
3o Other features and advantages of the invention will be apparent from the
following description of the preferred embodiments thereof, and from the
claims.
BRIEF DESCRIPTION OF TH,~'a,D~4vWINGS
CA 02335649 2001-O1-19
WO 00/04194 34 PCT/US99/16440
Figure 1 is a diagram showing the relationships of enzymes involved in 5-
FU metabolism and inhibition of thymidylate formation. Enzymes: 1. uridine
phosphorylase; 2. thymidine phosphorylase; 3. orotate phosphoribosyl
transferase; 4.
thymidine kinase; 5. uridine kinase; 6. ribonucletide reductase; 7.
thymidylate
synthase; 8. dCMP deaminase; 9. nucleoside monophosphate kinase; 10.
nucleoside
diphosphate kinase; 11. nucleoside diphosphatase or cytidylate kinase; 12:
thymine
phosphorylase. FH2 = dihydrofolate, FH4 = tetrahydrofolate. The Figure is
adapted
from Goodman & Gilman's The Pharmacological Basis of Therapeutics, ninth
edition, McGraw Hill, 1996, p. 1249.
Figure 2 is a diagram showing the relationship of enzymes related to folate
metabolism and formation of 5,10-methylenetetrahydrofolate. Enzymes: 1.
Fonminino-tetrahydrofolate cyclodeaminase; 2. methenyltetrahydrofolate
synthetase;
3. methenyltetra-hydrofolate cyclohydrolase; 4. formyltetrahydrofolate
synthetase; 5.
is formyltetrahydrofolate hydrolase; 6. formyltetrahydrofolate dehydrogenase;
7.
methyleneltetrahydrofolate dehydrogenase; 8. methyleneltetrahydrofolate
reductase
(MTHFR); 9. homocysteine methyltransferase (also called methionine
synthetase);
10. serine transhydroxymethylase; 11. glycine cleavage system; 12. thymidylate
synthase; 13. dihydrofolate reductase. Abbreviations: THF = tetrahydrofolate;
DHF
2o = dihydrofolate. Note that THF appears twice (i.e. the product of step 6 is
also
substrate for enzymes 10 and 11. Step 12 also appears in Figure 1, above. This
Figure is adapted from Mathews & van Holde, Biochemistry, The
Benjamin/Cummings Publishing Co., Redwood City CA, 1990, page 697.
25 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is generally described below in connection with cancer
chemotherapy. However, the described approach and techniques are applicable to
a
variety of other treatments and to genes associated with the efficacy and
safety of
30 such other treatments, for example, genes function in the pathways
identified below,
along with the specific genes listed. The present invention identifies a
number of
genes in certain treatment-related pathways, and further identifies a number
of
genetic sequence variances in those genes. The present description further
describes
how to identify variances which correlate with variable treatment efficacy and
35 further how to identify additional variances in the identified genes and
how to
determine the treatment response correlation of those additional variances.
Chemotherapy of cancer currently involves use of highly toxic drugs with
narrow therapeutic indices. Although progress has been made in the
CA 02335649 2001-O1-19
WO 00/04194 35 PCT/US99/16440
chemotherapeutic treatment of selected malignancies, most adult solid cancers
remain highly refractory to treatment. Nonetheless, chemotherapy is the
standard of
care for most disseminated solid cancers. Chemotherapy often results in a
significant
fraction of treated patients suffering unpleasant or life-threatening side
effects while
receiving little or no clinical benefit; other patients may suffer few side
effects
and/or have complete remission or even cure. Any test that could predict
response to
chemotherapy, even partially, would allow more selective use of toxic drugs,
and
could thereby significantly improve efficacy of oncologic drug use, with the
potential to both reduce side effects and increase the fraction of responders.
1o Chemotherapy is also expensive, not just because the drugs are often
costly, but also
because administering highly toxic drugs requires close monitoring by
carefully
trained personnel, and because hospitalization is often required for treatment
of (or
monitoring for) toxic drug reactions. Information that would allow patients to
be
divided into likely responder vs. non-responder (or likely side effect)
groups, with
only the former to receive treatment, would therefore also have a significant
impact
on the economics of cancer drug use.
Predicting Response to Chemotherapy
Several methods for predicting response to chemotherapy in individual
2o patients have been investigated over the years, ranging from the use of
biochemical
markers to testing drugs on a patient's cultured tumor cells. None of these
methods
has proven sufficiently informative and practical to gain wide acceptance.
However,
there are some specific examples of tests useful for predicting toxicity. For
example,
a diagnostic test to predict side effects associated with the antineoplastic
drugs 6-
2s mercaptopurine, 6-thioguanine and azathioprine has begun to gain wide
acceptance,
particularly among pediatric oncologists. Severe toxicity of thiopurine drugs
is
associated with deficiency of the enzyme thiopurine methyltransferase (TPMT).
Currently most TPMT testing is done using an enzyme assay, however the TPMT
gene has been cloned and mutations associated with low TPMT levels have been
3o identified; genetic testing is beginning to supplant enzyme assays because
genetic
tests are more easily standardized and economical.
While there are no good tests that predict positive chemotherapeutic
response, there is demonstrated utility to measuring estrogen and progesterone
receptor levels in cancer tissue before selecting therapy directed at
modulating
3s hormonal state. Measuring genetic variation in proteins that mediate the
effects,
course, outcome, and/or development of adverse events in those patients
potentially
receiving chemotherapy drugs is, in some respects, analogous to measuring ER
and
PR levels, which mediate the effects of hormones.
CA 02335649 2001-O1-19
WO 00/04194 36 PCT/US99/16440-
I. Outline: Identification of interpatient variation in response;
identification of
genes and variances relevant to drug action; development of diagnostic tests;
and use of variance status to determine treatment
Human therapeutic development follows a course from discovery and analysis in
a laboratory (preclinical development) to testing the candidate therapeutic
intervention in
human subjects (clinical development). The preclinical development of
candidate
therapeutic interventions for use in the treatment of human disease,
disorders, or
conditions begins at the discovery stage whereby a candidate therapy is tested
in vitro to
1o achieve a desired biochemical alteration of a biochemical or physiological
event. If
successful, the candidate is generally tested in animals to determine
toxicity, adsorption,
distribution, and metabolism within a living species. Occasionally, thane are
available
animal models that mimic human diseases, disorders, and conditions in which
testing the
candidate therapeutic intervention can pmvide supportive data to warrant
proceeding to
test the agent or compound in humans. When an agent or compound enters first
in human
studies, it is recognized that the prediction of whether the agent or
product's preclinical
success will be mimicked in humans is imperfect. Both safety and efficacy data
will
generally have to ultimately be determined in humans. Therefore, given
economic
constraints, and considering the complexities of human clinical trials, any
technical
2o advance to assist those skilled in the art of drug development will be
welcomed.
Advances can be implemented by aiding identification of genetic markers
associated with
interpatient variation in response during preclinical development (thereby
allowing
development of non-allele selective agents), or by identification or
optimization of
clinical trial design parameters in order to achieve successful development of
therapeutic
products at any stage of clinical development, or by identifying variables
that will allow
safe and efficacious use of a marketed product. Such advances will provide
benefits in
the form of therapeutic alternatives to those patients in need of medical
care.
As indicated in the Summary above, certain aspects of the present invention
typically involve the following process, which need not occur separately or in
the order
3o stated. Not all of these described processes must be present in a
particular method, or
need be performed by a single entity or organization or person. Additionally,
if certain of
the information is available from other sources, that information can be
utilized in the
present invention. The processes are as follows: a) variability between
patients in the
response to a particular treatment is observed; b) at least a portion of the
variable
response is correlated with the presence or absence of at least one variance
in at least one
gene; c) an analytical or diagnostic test is provided to determine the
presence or absence
of the at least one variance in individual patients; d) the presence or
absence of the
CA 02335649 2001-O1-19
WO 00/04194 3~ PCT/US99/16440
variance or variances is used to select a patient for a treatment.or to select
a treatment for
a patient, or the variance information is used in other methods described
herein.
A. Identification of Interpatient Variability in Response to a Treatment
Inteipatient variability is the rule, not the exception, in clinical
therapeutics. One
of the best sources of information on interpatient variability is the nurses
and physicians
supervising the clinical trial who accumulate a body of first hand
observations of
physiological responses to the drug in different normal subjects or patients.
Evidence of
interpatient variation in response can also be measured statistically, and may
be best
to described by statistical measures that examine magnitude of response
(beneficial or
adverse) across a large number of subjects.
In accord with the other portions of this description, the present invention
concerns DNA sequence variances that can affect one or more of
i. The susceptibility of individuals to a disease;
ii. The course or natural history of a disease;
iii. The response of a patient with a disease to a medical intervention, such
as, for
example, a drug, a biologic substance, physical energy such as radiation
therapy, or a
specific dietary regimen. The ability to predict either beneficial or
detrimental responses
is medically useful.
2o Thus variation in any of these three parameters may constitute the basis
for
initiating a pharmacogenetic study directed to the identification of the
genetic sources of
interpatient variation. The effect of a DNA sequence variance or variances on
disease
susceptibility or natural history (i and ii, above) are of particular interest
as the variances
can be used to define patient subsets which behave differently in response to
medical
interventions such as those described in (iii).
In other words, a variance can be useful for customizing medical therapy at
least
for either of two reasons. First, the variance may be associated with a
specific disease
subset that behaves differently with respect to one or more therapeutic
interventions (i
and ii above); second, the variance may affect response to a specific
therapeutic
intervention (iii above). Consider for exemplary purposes pharmacological
therapeutic
interventions. In the first case, there may be no effect of a particular gene
sequence
variance on the observable pharmacological action of a drug, yet the disease
subsets
defined by the variance or variances differ in their response to the drug
because, for
example, the drug acts on a pathway that is more relevant to disease
pathophysiology in
one variance-defined patient subset thanin another variance-defined patient
subset. The
second type of useful gene sequence variance affects the pharmacological
action of a drug
or other treatment. Effects on pharmacological responses fall generally into
two
categories; phannacokinetic and pharmacodynamic effects. These effects have
been
CA 02335649 2001-O1-19
WO 00/04194 38 PCT/US99/16440
defined as follows in Goodman and Gilman's Phamacologic Basis of Therapeutics
(ninth
edition, McGraw Hill, New York, 1986): "Pharmacokinetics" deals with the
absorption,
distribution, biotransformations and excretion of drugs. The study of the
biochemical and
physiological effects of drugs and their mechanisms of action is termed
" pharmacodynamics."
Useful gene sequence variances for this invention can be described as
variances
which partition patients into two or more groups that respond differently to a
therapy,
regardless of the reason for the difference, and regardless of whether the
reason for the
difference is known.
to
B. Identification of Specific Genes and Correiation of Variances in Those
Genes
with Response to Treatment of Diseases or Conditions
It is useful to identify particular genes which do or are likely to mediate
the
efficacy or safety of a treatment method for a disease or condition,
particularly in view of
t5 the large number of genes which have been identified and which continue to
be identified
in humans. As is further discussed in section C below, this correlation can
proceed by
different paths. One exemplary method utilizes prior information on the
pharmacology or
pharmacokinetics or pharmacodynamics of a treatment method, e.g., the action
of a drug,
which indicates that a particular gene is, or is likely to be, involved in the
action of the
20 treatment method, and further suggests that variances in the gene may
contribute to
variable response to the treatment method.
Alternatively, if such information is not known, variances in a gene can be
correlated empirically with treatment response. In this method, variances in a
gene which
exist in a population can be identified. The presence of the different
variances or
i5 haplotypes in individuals of a study group, which is preferably
representative of a
population or populations, is determined. This variance information is then
correlated
with treatment response of the various individuals as an indication that
genetic variability
in the gene is at least partially responsible for differential treatment
response. Statistical
measures known to those skilled in the art are preferably used to measure the
fraction of
3o interpatient variation attributable to any one variance.
Useful methods for identifying genes relevant to the physiologic action of a
drug
or other treatment are known to those skilled in the art, and include large
scale analysis of
gene expression in cells treated with the drug compared to control cells, or
large scale
analysis of the protein expression pattern in treated vs. untreated cells, or
the use of
35 techniques for identification of interacting proteins or ligand-protein
interactions.
C. Development of a Diagnostic Test to Determine Variance Status
CA 02335649 2001-O1-19
WO 00/04194 39 PCT/US99/16440
In accordance with the description in the Summary above, the present invention
generally concerns the identification of variances in genes which are
indicative of the
effectiveness of a treatment in a patient. The identification of specific
variances, in
effect, can be used as a diagnostic or prognostic test. Correlation of
treatment efficacy
and/or toxicity with particular genes and gene families or pathways is
provided in
Stanton et al., U.S. Provisional Application 601093,484, filed July 20, 1998,
entitled
GENE SEQUENCE VARIANCES WITH UTILITY IN DETERMINING THE
TREATMENT OF DISEASE (concerns the safety and efficacy of compounds active on
folate or pyrimidine metabolism or action).
io Genes identified in the examples below and the attached Tables and Figures
can
be used in the present invention.
Methods for diagnostic tests are well known in the art. Generally in this
invention, the diagnostic test involves determining whether an individual has
a variance
or variant form of a gene that is involved in the disease or condition or the
action of the
is drug or other treatment or effects of such treatment. Such a variance or
variant form of
the gene is preferably one of several different variances or forms of the gene
that have
been identified within the population and are known to be present at a certain
frequency.
In an exemplary method, the diagnostic test involves performed by amplifying a
segment
of DNA or RNA (generally after converting the RNA to cDNA) spanning one or
more
2o variances in the gene sequence. Preferably, the amplified segment is <500
bases in
length, in an alternative embodiment the amplified segment is <100 bases in
length, most
preferably <45 bases in length. In many cases, the diagnostic test is
performed by
amplifying a segment of DNA or RNA (cDNA) spanning a variance, or even
spanning
more than one variance in the gene sequence and preferably maintaining the
phase of the
25 variances on each allele. The term "phase" means the association of
variances on a single
copy of the gene, such as the copy transmitted from the mother (maternal copy
or
maternal allele) or the father (paternal copy or paternal allele). It is
apparent that such
diagnostic tests are performed after initial identification of variances
within the gene.
Diagnostic genetic tests useful for practicing this invention belong to two
types:
3o genotyping tests and haplotyping tests. A genotyping test simply provides
the status of a
variance or variances in a subject or patient. For example suppose nucleotide
150 of
hypothetical gene X on an autosomal chromosome is an adenine (A) or a guanine
(G)
base. The possible genotypes in any individual are AA, AG or GG at nucleotide
150 of
gene X.
35 In a haplotyping test there is at least one additional variance in gene X,
say at
nucleotide 810, which varies in the population as cytosine (C) or thymine (T).
Thus a
particular copy of gene X may have any of the following combinations of
nucleotides at
positions 150 and 810: 150A-810C, 150A-810T, 1506-810C or 1506-810T. Each of
the
CA 02335649 2001-O1-19
WO 00/04194 ~ PCT/US99/1b440
four possibilities is a unique haplotype. If the two nucleotides interact in
either RNA or
protein, then knowing the haplotype can be important. The point of a
haplotyping test is
to determine the haplotypes present in a DNA or cDNA sample (e.g. from a
patient). In
the example provided there are only four possible haplotypes, but, depending
on the
number of variances in the gene and their distribution in human populations
there may be
three, four, five, six or more haplotypes at a given gene. The most useful
haplotypes for
this invention are those which occur commonly in the population being treated
for a
disease or condition. Preferably such haplotypes occur in at least 5% of the
population,
more preferably in at least 10%, still more preferably in at least 20% of the
population
to and most preferably in at least 30% or more of the population. Conversely,
when the goal
of a pharmacogenetic program is to identify a relatively rare population that
has an
adverse reaction to a treatment, the most useful haplotypes may be rare
haplotypes, which
may occur in less than 5%, less than 2%, or even in less than 1 % of the
population. One
skilled in the art will recognize that the frequency of the adverse reaction
will provide a
t 5 useful guide to the likely frequency of salient causative haplotypes.
Based on the identification of variances or variant forms of a gene, a
diagnostic
test utilizing methods known in the art can be used to determine whether a
particular form
of the gene, containing specific variances or haplotypes, or combinations of
variances and
haplotypes, is present in at least one copy, one copy, or more than one copy
in an
20 individual. Such tests are commonly performed using DNA or RNA collected
firm
blood, cells, tissue scrapings or other cellular materials, and can be
performed by a
variety of methods including, but not limited to, hybridization with allele-
specific probes,
enzymatic mutation detection, chemical cleavage of mismatches, mass
spectrometry or
DNA sequencing, including minisequencing. Methods for haplotyping are provided
in
2s this application. In particular embodiments, hybridization with allele
specific probes can
be conducted in two formats: (1) allele specific oligonucleotides bound to a
solid phase
(glass, silicon, nylon membranes) and the labelled sample in solution, as in
many DNA
chip applications, or (2) bound sample (often cloned DNA or PCR amplified DNA)
and
labelled oligonucleotides in solution (either allele specific or short so as
to allow
3o sequencing by hybridization). The application of such diagnostic tests is
possible after
identification of variances that occur in the population. Diagnostic tests may
involve a
panel of variances from one or more genes, often on a solid support, which
enables the
simultaneous determination of more than one variance in one or more genes.
D. Use of Variance Status to Determine Treatment
35 The present disclosure describes exemplary gene sequence variances in genes
identified in a gene table herein (e.g., Tables 2, 6, and 8), and variant
forms of these gene
that may be determined using diagnostic tests. As indicated in the Summary,
such a
variance-based diagnostic test can be used to determine whether or not to
administer a
CA 02335649 2001-O1-19
PCTNS99/16440
W O 00/04194 41
specific drug or other treatment to a patient for treatment of a disease or
condition.
Preferably such diagnostic tests are incorporated in texts such as Clinical
Diagnosis and
Management by Laboratory Methods ( 19th Ed) by John B. Henry (Editor) W B
Saunders
Company, 1996; Clinical Laboratory Medicine : Clinical Application of
Laboratory Data,
(6th edition) by R. Ravel, Mosby-Year Book, 1995, , or medical textbooks
including,
without limitation, textbooks of medicine, laboratory medicine, therapeutics,
pharmacy,
pharmacology, nutrition, allopathic, homeopathic, and osteopathic medicine;
most
preferably such a diagnostic test is specified by regulatory authorities,
e.g., by the U.S.
Food and Drug Administration, and is incorporated in the label or insert as
well as the
to Physicians Desk Reference.
In such cases, the procedure for using the drug is restricted or limited on
the basis
of a diagnostic test for determining the presence of a variance or variant
form of a gene.
The procedure may include the route of administration of the drug, the dosage
form,
dosage, schedule of administration or use with other drugs; any or all of
these may
is require selecting or determination consistent with the results of the
diagnostic test or a
plurality of such tests. Preferably the use of such diagnostic tests to
determine the
procedure for administration of a drug is incorporated in a text such as those
listed above,
or medical textbooks, for example, textbooks of medicine, laboratory medicine,
therapeutics, pharmacy, pharmacology, nutrition, allopathic, homeopathic, and
20 osteopathic medicine. As previously stated, preferably such a diagnostic
test or tests are
required by regulatory authorities and are incorporated in the label or insert
as well as the
Physicians Desk Reference.
Variances and variant forms of genes useful in conjunction with treatment
methods may be associated with the origin or the pathogenesis of a disease or
condition.
2s In many useful cases, the variant form of the gene is associated with a
specific
characteristic of the disease or condition that is the target of a treatment,
most preferably
response to specific drugs or other treatments. Examples of diseases or
conditions
ameliorable by the methods of this invention are identified in the Examples
and tables
below; in general treatment of disease with current methods, particularly drug
treatment,
3o always involves some unknown element (involving efficacy or toxicity or
both) that can
be reduced by appropriate diagnostic methods.
Alternatively, the gene is involved in drug action, and the variant fonms of
the
gene are associated with variability in the action of the drug. For example,
in some cases,
one variant form of the gene is associated with the action of the drug such
that the drug
35 will be effective in an individual who inherits one or two copies of that
foam of the gene.
Alternatively, a variant form of the gene is associated with the action of the
drug such
that the drug will be toxic or otherwise contra-indicated in an individual who
inherits one
or two copies of that form of the gene.
CA 02335649 2001-O1-19
PCTNS99I16440
WO 00/04194 42
In accord with this invention, diagnostic tests for variances and variant
forms of
genes as described above can be used in clinical trials to demonstrate the
safety and
efficacy of a drug in a specific population. As a result, in the case of drugs
which show
variability in patient response correlated with the presence or absence of a
variance or
variances, it is preferable that such drug is approved for sale or use by
regulatory agencies
with the recommendation or requirement that a diagnostic test be performed for
a specific
variance or variant form of a gene which identifies specific populations in
which the drug
will be safe and/or effective. For example, the drug may be approved for sale
or use by
regulatory agencies with the specification that a diagnostic test be performed
for a
to specific variance or variant form of a gene which identifies specific
populations in which
the drug will be toxic. Thus, approved use of the drug, or the procedure for
use of the
drug, can be limited by a diagnostic test for such variances or variant forms
of a gene; or
such a diagnostic test may be considered good medical practice, but not
absolutely
required for use of the drug.
is As indicated, diagnostic tests for variances as described in this invention
may be
used in clinical trials to establish the safety and efficacy of a drug.
Methods for such
clinical trials are described below and/or are known in the art and are
described in
standard textbooks. For example, diagnostic tests for a specific variance or
variant form
of a gene may be incorporated in the clinical trial protocol as inclusion or
exclusion
20 criteria for enrollment in the trial, to allocate certain patients to
treatment or control
groups within the clinical trial or to assign patients to different treatment
cohorts.
Alternatively, diagnostic tests for specific variances may be performed on all
patients
within a clinical trial, and statistical analysis performed comparing and
contrasting the
efficacy or safety of a drug between individuals with different variances or
variant forms
25 of the gene or genes. Preferred embodiments involving clinical trials
include the genetic
stratification strategies, phases, statistical analyses, sizes, and other
parameters as
described herein.
Similarly, diagnostic tests for variances can be performed on groups of
patients
known to have efficacious responses to the drug to identify differences in the
frequency
3o of variances between responders and non-responders. Likewise, in other
cases,
diagnostic tests for variance are performed on groups of patients known to
have toxic
responses to the drug to identify differences in the frequency of the variance
between
those having adverse events and those not having adverse events. Such outlier
analyses
may be particularly useful if a limited number of patient samples are
available for
35 analysis. It is apparent that such clinical trials can be or are performed
after identifying
specific variances or variant forms of the gene in the population.
The identification and confirmation of genetic variances is described in
certain
patents and patent applications. The description therein is useful in the
identification of
CA 02335649 2001-O1-19
PCT/US99/16440
WO 00/04194 43
variances in the present invention. For example, a strategy for the
development of
anticancer agents having a high therapeutic index is described in Housman,
International Application PCT/US/94 08473 and Housman, INHIBITORS OF
ALTERNATIVE ALLELES OF GENES ENCODING PROTEINS VITAL FOR CELL
s VIABILITY OR CELL GROWTH AS A BASIS FOR CANCER THERAPEUTIC
AGENTS, U.S. Patent 5,702,890, issued December 30, 1997, which are hereby
incorporated by reference in their entireties. Also, a number of gene targets
and
associated variances are identified in Housman et al., U.S. Patent Application
09/045,053, entitled TARGET ALLELES FOR ALLELE-SPECIFIC DRUGS, filed
1o March i9, 1998, which is hereby incorporated by reference in its entirety,
including
drawings.
The described approach and techniques are applicable to a variety of other
diseases, conditions, and/or treatments and to genes associated with the
etiology and
pathogenesis of such other diseases and conditions and the efficacy and safety
of such
15 other treatments.
Useful variances for this invention can be described generally as variances
which
partition patients into two or more groups that respond differently to a
therapy (a
therapeutic intervention), regardless of the reason for the difference, and
regardless of
whether the reason for the difference is known.
II. From Variance List to Clinical Trlal: Identifying Genes and Gene
Variances that Account for Variable Responses to Treatment
There are a variety of useful methods for identifying a subset of genes firm a
large set that should be prioritized for further investigation with respect to
their influence
on inter-individual variation in disease predisposition or response to a
particular drug.
These methods include for example, (1) searching the relevant literature to
identify genes
relevant to a disease or the action of a drug; (2) screening the genes
identified in step 1
for variances. A large set of exemplary variances are provided in Tables 3, 4,
and 10; (3)
using computational tools to predict the functional effects of variances in
specific genes;
(4) using in vitro or in vivo experiments to identify genes which may
participate in the
response to a drug or treatment, and to determine the variances which affect
gene, RNA
or protein fimction, and may therefore be important genetic variables
affecting disease
manifestations or drug response; and (5) retrospective or prospective clinical
trials. Each
of these methods is considered below in some detail.
(1) To begin, one preferably identifies, for a given treatment, a set of
candidate genes that
are likely to affect disease phenotype or drug response. This can be
accomplished
most efficiently by first assembling the relevant medical, pharmacological and
CA 02335649 2001-O1-19
PCT/US99/16440
W O 00/04194 ~ -
biological data from available sources (e.g., public databases and
publications). One
skilled in the art can review the literature (textbooks, monographs, journal
articles)
and online sources {databases) to identify genes most relevant to the action
of a
specific drug or other treatment, particularly with respect to its utility for
treating a
s specific disease, as this beneficially allows the set of genes to be
analyzed ultimately
in clinical trials to be reduced from an initial large set. Specific
strategies for
conducting such searches are described below. In some instances the literature
may
provide adequate information to select genes to be studied in a clinical
trial, but in
other cases additional experimental investigations of the sort described below
will be
1 o preferable to maximize the likelihood that the salient genes and variances
are moved
forward into clinical studies. Experimental data are also useful in
establishing a list of
candidate genes, as described below.
(2) Having assembled a list of candidate genes generally the second step is to
screen for
variances in each candidate gene. Experimental and computational methods for
15 variance detection are described in this invention, and a tables of
exemplary variances
is provided (e.g., Table 3, 4, or 10) as well as methods for identifying
additional
vanances.
(3) Having identified variances in candidate genes the next step is to assess
their likely
contribution to clinical variation in patient response to therapy, preferably
by using
2o informatics-based approaches such as DNA and protein sequence analysis and
protein
modeling. The literature and informatics-based approaches provide the basis
for
prioritization of candidate genes, however it may in some cases be desirable
to further
narrow the list of candidate genes, or to measure experimentally the phenotype
associated with specific variances or sets of variances (e.g. haplotypes).
25 (4) Thus, as a third step in candidate gene analysis, one skilled in the
art may elect to
perform in vitro or in vivo experiments to assess the functional importance of
gene
variances, using either biochemical or genetic tests. (Certain kinds of
experiments -
for example gene expression profiling and proteome analysis - may not only
allow
refinement of a candidate gene list but may also lead to identification of
additional
3o candidate genes.) Combination of two or all of the three above methods will
provide
sufficient information to narrow the set of candidate genes and variances to a
number
that can be studied in a clinical trial with adequate statistical power.
(5) The fourth step is to design retrospective or prospective human clinical
trials to test
whether the identified allelic variance, variances, or haplotypes or
combination
35 thereof influence the efficacy or toxicity profiles for a given drug or
other therapeutic
intervention. It should be recognized that this fourth step is the crucial
step in
producing the type of data that would justify introducing a diagnostic test
for at least
one variance into clinical use. Thus while each of the above four steps are
useful in
CA 02335649 2001-O1-19
WO 00/04194 PCTNS99/16440
particular instances of the invention, this final step is indispensable.
Further guidance
and examples of how to perform these five steps is provided below.
1. Identification of Candidate Genes Relevant to the Action of a Drug
s Practice of this invention will often begin with identification of a
specific
pharmaceutical product, for example a drug, that would benefit from improved
efficacy
or reduced toxicity or both, and the recognition that pharmacogenetic
investigations as
described herein provide a basis for achieving such improved characteristics.
The
question then becomes which of the genes and variances provided in this
application, e.g.,
to in Tables 3, 4, and 10, would be most relevant to interpatient variation in
response to the
drug. As discussed above, the set of relevant genes includes both genes
involved in the
disease process and genes involved in the interaction of the patient and the
treatment - for
example genes involved in pharmacokinetic and pharmacodynamic action of a
drug. The
biological and biomedical literature and online databases provide useful
guidance in
15 selecting such genes. Specific guidance in the use of these resources is
provided below.
Review the literature and online sources
One way to find genes that affect response to a drug in a particular disease
setting
is to review the published literature and available online databases regarding
the
2o pathophysiology of the disease and the pharmacology of the drug. Literature
or online
sources can provide specific genes involved in the disease process or drug
response, or
describe biochemical pathways involving multiple genes, each of which may
affect the
disease process or drug response.
Alternatively, biochemical or pathological changes characteristic of the
disease
25 may be described; such information can be used by one skilled in the art to
infer a set of
genes that can account for the biochemical or pathologic changes. For example,
to
understand variation in response to a drug that modulates serotonin levels in
a central
nervous system (CNS) disorder associated with altered levels of serotonin one
would
preferably study, at a minimum, variances in genes responsible for serotonin
3o biosynthesis, release from the cell, receptor binding, presynaptic
reuptake, and
degradation or metabolism. Genes responsible for each of these fiuictions
should be
examined for variation that may account for interpatient differences in drug
response or
disease manifestations. As recognized by those skilled in the art, a
comprehensive list of
such genes can be obtained from textbooks, monographs and the literature.
35 There are several types of scientific information, described in some detail
below,
that are valuable for identifying a set of candidate genes to be investigated
with respect to
a specific disease and therapeutic intervention. First there is the medical
literature, which
provides basic information on disease pathophysiology and therapeutic
interventions. A
CA 02335649 2001-O1-19
46
WO 00/04194 PCT/US99/16440
subset of this literature is devoted to specific description of pathologic
conditions.
Second there is the pharmacology literature, which will provide additional
information on
the mechanism of action of a drug (pharmacodynamics) as well as its principal
routes of
metabolic transformation (pharmacokinetics) and the responsible proteins.
Third there is
the biomedical literature (principally genetics, physiology, biochemistry and
molecular
biology), which provides more detailed information on metabolic pathways,
protein
structure and function and gene structure. Fourth, there are a variety of
online databases
that provide additional information on metabolic pathways, gene families,
protein
function and other subjects relevant to selecting a set of genes that are
likely to affect the
1o response to a treatment.
Medical Literature
A good starting place for information on molecular pathophysiology of a
specific
disease is a general medical textbook such as Ii,~rrison's Pri~cinles of
Internal Medicine.
15 14th edition; (2 Vol Set) by A.S. Fauci, E. Braunwald, K.J. Isselbacher, et
al. (editors),
McGraw Hill, 1997, or Cecil Textbook of Medicine (20th Ed) by R. L. Cecil, F.
Plum
and J. C. Bennett (Editors) W B Saunders Co., 1996. For pediatric diseases
texts such as
Nelson Textbook of Pediatrics (15th edition) by R.E. Behrman, R.M. Kliegman,
A.M.
Arvin and W.E. Nelson (Editors), W B Saunders Co., 1995 or Os~i's Princi
Ie~Zs_an_d
2o Practice of Pe iatrics (3'~ Edition) by J.A. Mamillan & F.A. Oski
Lippincott-Raven, 1999
are useful introductions. For obstetrical and gynecological disorders texts
such as
Willi,~ns Obstetrics (20th Ed) by F.G. Cunningham, N.F. Gant, P.C. McDonald et
al.
(Editors), Appleton & Lange, 1997 provide general information on disease
pathophysiology. For psychiatric disorders texts such as the ~~prehensive
Textbook of
25 Ps3rchi~,y, VI (2 Vols) by H.I. Kaplan and B.J. Sadock (Editors),
Lippincott, Williams &
Wilkins, 1995, or ~ ~A_m~can Psychiatric Press Textbook of Psvchia (3'~
edition) by
R.E. Hales, S.C. Yudofsky and J.A. Talbott (Editors) Amer Psychiatric Press,
1999
provide an overview of disease nosology, pathophysiological mechanisms and
treatment
regunens.
3o In addition to these general texts, there are a variety of more specialized
medical
texts that provide greater detail about specif c disorders which can be
utilized in
developing a list of candidate genes and variances relevant to interpatient
variation in
response to a treatment. For example, within the field of medicine there are
standard
textbooks for each of the subspecialties. Some specific examples include:
35 Heart Disease' A Textbook of Cardiovascular Medicine (2 Volume set) by E.
Braunwald (Editor), W B Saunders Co., 1996.
H~,~~ the arc. Arteries and Veins (9th Ed) (2 Vol Set) by R.W. Alexander,
R.C. Schlant, V. Fuster, W. Alexander and E.H. Sonnenblick (Editors) McGraw
Hill,
CA 02335649 2001-O1-19
PCT/US99/16440
WO 00/04194 47 -
1998.
Princi~ es of NeuroloQV (6th edition) by R.D. Adams, M. Victor (editors), and
A.H. Ropper (Contributor), McGraw Hill, 1996.
SIeiseneer &. Fordtra~n's Gastrointestinal and Liver Disease' Pathoph sy
ioloQv
Diagnosis. Manaeement (6th edition) by M. Feldman, B.F. Scharschmidt and M.
Sleisenger (Editors), W B Saunders Co., 1997.
Textbook of Rheumatoloev (5th edition) by W.N. Kelley, S. Ruddy, E.D. Hands
Jr. and C.B. Sledge (Editors) (2 volume set) W B Saunders Co., 1997.
Williams Textbook of EndocrinoloQV (9th edition) by J.D. Wilson, D.W. Foster,
to H. M. Kronenberg and Larsen (Editors), W B Saunders Co., 1998.
Wintrobe's Clinical Hem tolosv (10th Ed) by G.R. Lee, J. Foerster {Editor) and
J.
Lukens (Editors) (2 Volumes) Lippincott, Williams & Wilkins, 1998.
~ancer~ Principles & Practice of Oncology (5th edition) by V.T. Devita, S.A.
Rosenberg and S. Hellman (editors), Lippincott-Raven Publishers, 1997.
p_ rinclples of Pulmonary Medicine (3rd edition) by S.E. Weinberger & J
Fletcher
(Editors), W B Saunders Co., 1998.
pj,~enosis and Man~gem t of Renal Disease and Hypertension (2nd edition) by
A.K.
Mandal & J.C. Jennette (Editors), Carolina Academic Press, 1994.Massrv &
Glassock's
T, xtbook of Ne~hroloev (3rd edition) by S.G. Massry & R.J. Glassock (editors)
Williams
& Wilkins, 1995.
The Management of Pain by J.J. Bonica, Lea and Febiger, 1992
Oohthalmolosv by M. Yanoff & J.S. Duker, Mosby Year Book, 1998
~t~nical hthalmoloev: A Sy,~ emic Aur~roach by J.J. Kanski, Butterworth-
Heineman,
1994.Essential Otolarvng,~loev by J.K. Lee Appleton and Lange 1998.
2s
In addition to these subspecialty texts there are many textbooks and
monographs
that concern more restricted disease areas, or specific diseases. Such books
provide more
extensive coverage of pathophysiologic mechanisms and therapeutic options. The
number of such books is too great to provide examples for all but a few
diseases, however
one skilled in the art will be able to readily identify relevant texts. One
simple way to
search for relevant titles is to use the search engine of an online bookseller
such as
h~g://www.amazon.com or ~tp~//www.barnesandnoble.com using the disease or drug
(or
the group of diseases or drugs to which they belong) as search terms. For
example a
search for asthma would turn up titles such as Asthma ~ Basic Mechanisms and
Clinical
Manaeement (3rd edition) by P.J. Barnes, LW. Rodger and N.C. Thomson
(Editors),
Academic Press, 1998 and Airways and Vascular Remodelling in Asthma and
Cardiovascular Disease : implications for Therapeutic Intervention : Based on
the
Scientific Program, by C. Page & J. Black (Editors), Academic Press, 1994.
CA 02335649 2001-O1-19
PCT/US99/16440
wo ooio4i94 48 -
Pathology Literature
In addition to medical texts there are texts that specifically address disease
etiology and pathologic changes associated with disease. A good general
pathology text
is Robbins Patholog'~c Basis of Disease (6th edition) by R.S. Cotran, V.
Kumar, T.
Collins and S.L. Robbins, W B Saunders Co., 1998. Specialized pathology texts
exist for
each organ system and for specific diseases, similar to medical texts. These
texts are
useful sources of information for one skilled in the art for developing lists
of genes that
may account for some of the known pathologic changes in disease tissue.
Exemplary
texts are as follows:
to Bone Marrow Patholoav 2"° edition, by B.J. Bain, I. Lampert. & D.
Clark, Blackwell
Science,1996
Atlas of Renal Pa holoev by F.G. Silva, W.B. Saunders, 1999.
Fundamentals of Toxicol2g~ tholoev by W.M. Haschek and C.G. Rousseaux,
Academic Press, 1997.
15 troin gstinal Pathoiosv by P. Chandrasoma, Appleton and Lange, 1998.
Q~ht_halmic Patholog~r with Clinical Correlations by J. Sassani, Lippincott-
Raven, 1997.
Pathology of Bone an 3oint Disorders by F. McCarthy, F.J. Frassica and A.
Ross, W. B.
Saunders, 1998.
~~~~;~ Patholoev by M.A. Grippi, Lippicott-Raven, 1995.
2o Neurovatholosv by D. Ellison, L. Chimelli, B. Handing, S. Love& J. Lowe,
Mosby Year
Book, 1997.
rreenfield's NeurovatholQV 6'" edition by J.G. Greenfield, P.L. Lantos & D.I.
Graham,
Edward Arnold, 1997.
25 Pharmacology, Pharmacogenetics and Pharmacy Literature
There are also both general and specialized texts and monographs on
pharmacology that provide data on pharmacokinetics and pharmacodynamics of
drugs.
The discussion of pharmacodynamics (mechanism of action of the drug)in such
texts is
often supported by a review of the biochemical pathway or pathways that are
affected by
3o the drug. Also, proteins related to the target protein are often listed; it
is important to
account for variation in such proteins as the related proteins may be involved
in drug
pharmacology. For example, there are 14 known serotonin receptors. Various
pharmacological serotonin agonists or antagonists have different affinities
for these
different receptors. Variation in a specific receptor may affect the
pharmacology not only
35 of drugs intentionally targeted to that reccptor, but also drugs targeted
to different
receptors, that may have differential'~action on two allelic forms of the non-
targeted
receptor. Thus genes encoding proteins structurally related to the target
protein are useful
for screening for variance in the present invention. A good general
pharmacology text is
CA 02335649 2001-O1-19
49
WO 00/04194 PCTNS9911644t~
Good_ma_n & Gilma_n's thg Pha_~acolog~c_al Basis of'f~eraneutics (9th Ed) by
J.G.
Hardman, L.E. Limbird, P.B. Molinoff, R.W. Ruddon and A.G. Gilman (Editors)
McGraw Hill, 1996. There are also texts that focus on the pharmacology of
drugs for
specific disease areas, or specific classes of drugs (e.g. natural products)
or adverse drug
interactions, among other subjects. Specific examples include:
The American Psychiatric Press Textbook of Psvchonharmacologv (2nd edition)
by A.F. Schatzberg & C.B. Nemeroff (Editors), Amer Psychiatric Press, 1998.
ISBN:
0880488174
F~a~tial Psychonharm coloQV ~ Neuroscientific Basis and ~'~ractica
Appjications
1o by N. Muntner and S.M. Stahl, Cambridge Univ Press, 1996.
There are also texts on pharmacogenetics which are particularly useful for
identifying genes which may contribute to variable pharmacokinetic response.
In
addition there are texts on some of the major xenobiotic metabolizing
proteins, such as
the cytochrome P450 genes.
15 ~, ~a ogenetics of Drug Metabolism (International Encyclopedia of
Pharmacology and Therapeutics) by Werner Kalow (Editor) Pergamon Press, 1992.
~g~net~~ Fa~~~~ in Dru~py ' linical and Molecylar Pharmacog~enetics by
D.A Price Evans, Cambridge Univ Press, 1993.
Pharmacogenetics (Oxford Monographs on Medical Genetics, 32) by W.W.
2o Weber, Oxford Univ Press, 1997.
~w~~h_mme P450 ~ Structure Mechanism and Biochemistry by P.R. Ortiz de
Montellano (Editor), Plenum Publishing Corp, 1995.
Annleton & Lange's Review of Pharmacy, 6'" edition, (Appleton & Lange's
Review Series) by G.D. Hall & B.S. Reiss, Appleton & Lange, 1997.
Genetics, Biochemistry and Molecular Biology Literature
In addition to the medical, pathology, and pharmacology texts listed above
there
are several information sources that one skilled in the art will turn to for
information on
the genetic, physiologic, biochemical, and molecular biological aspects of the
disease,
3o disorder or condition or the effect of the therapeutic intervention on
specific physiologic
processes. The biomedical literature may include information on nonhuman
organisms
that is relevant to understanding the likely disease or pharmacological
pathways in man.
Genetic texts may provide insight into the likely effect of an allelic
variance,
variances, or haplotypes on individual responses to a therapeutic
intervention,
particularly if there are genetic variances known to effect drug response.
Example 1
describes variances in the dihydropyrimidine dehydrogenase (DPD) gene locus
and their
effects on fluoropyrimidine catabolism. DPD is an example of a gene that, in
rare mutant
forms, is associated with severe fluoropyrimidine poisoning. It is reasonable
to expect
CA 02335649 2001-O1-19
So
WO 00/04194 PCT/US99/16440
that more common alleles may exist at the DPD locus and may affect
fluoropyrimidine
metabolism, thus accounting for interpatient variation. Thus the genetics of a
rare allele
or alleles may provide a basis for examining the effects of commonly occuring
alleles on
moderate phenotypes. The genetics of rate DPD deficiency is well described in
medical
genetics textbooks listed below, for example see Scriver et al (full citation
below).
Also provided below are illustrative texts which will aid in the
identification of a
pathway or pathways, and a gene or genes that may be relevant to
interindividual
variation in response to a therapy. Textbooks of biochemistry, genetics and
physiology
are often useful sources for such pathway information. In order to ascertain
the
1o appropriate methods to analyze the effects of an alleleic variance,
variances, or
haplotypes in vitro, one skilled in the art will review existing information
on molecular
biology, cell biology, genetics, biochemistry; and physiology. Such texts are
useful
sources for general and specific information on the genetic and biochemical
processes
involved in disease and in drug action, as well as experimental procedures
that may be
useful in performing in vitro research on an allelic variance, variances, or
haplotye.
Texts on gene structure and function and RNA biochemistry will be useful in
evaluating the consequences of variances that do not change the coding
sequence. Such
variances may alter the interaction of RNA with proteins or other regulatory
molecules
affecting RNA processing, polyadenylation, and export.
Molecular and Cellular Biology
Molecular Cell Biology by H. Lodish, D. Baltimore, A. Berk, L. Zipurksy & J.
Darnell,
W H Freeman & Co., 1995.
"Essentials of Molecular Biology", D. Freifelder and MalacinskiJones and
Bartlett, 1993.
"Genes and Genomes: A Changing Perspective", M. Singer and P. Berg, 1991.
University
Science Books
"Gene Structure and Expression", J.D. Hawkins, 1996. Cambridge University
Press
Molecular Biology of the Cell, 2nd edition, B. Alberts et alGarland
Publishing, 1994.,
Molecular Genetics
The Metabolic and Molecular Bases of Inherited Disease by C. R. Scriver, A.L.
Beaudet,
W.S. Sly (Editors), 7th edition, McGraw Hill, 1995
"Genetics and Molecular Biology", R. Schleif, 1994. 2nd edition, Johns Hopkins
University Press
"Genetics", P.J. Russell, 1996. 4th edition, Harper Collies
"An Introduction to Genetic Analysis", Griffiths et a1.1993. 5th edition, W.H.
Freeman
and Company
"Understanding Genetics: A molecular approach", Rothwell, 1993. Wiley-Liss
CA 02335649 2001-O1-19
51
WO 00/04194 PCT/US99/16440
General Biochemistry
"Biochemistry", L. Stryer, 1995. W.H. Freeman and Company
"Biochemistry", D. Voet and J.G. Voet, 1995. John Wiley and Sons
"Principles of Biochemistry", A.L. Lehninger, D.L. Nelson, and M.M. Cox, 1993.
Worth
Publishers
"Biochemistry", G. Zubay, 1998. Wm. C. Brown Communications
"Biochemistry", C.K. Mathews and K.E. van Holde, 1990. Benjamin/Cummings
Transcription
"Eukaryotic Transcriptiuon Factors", D.S. Latchman, 1995. Academic Press
"Eukaryotic Gene Transcription", S. Goodboum (ed.), 1996. Oxford University
Press.
"Transcription Factors and DNA Replication", D.S. Pederson and N.H. Heintz,
1994.
CRC Press/R.G. Landes Company
"Transcriptional Regulation", S.L. McKnight and K. Yamamoto (eds.), 1992. 2
volumes,
Cold Spring Harbor Laboratory Press
RNA
"Control of Messenger RNA Stability", J. Belasco and G. Brawerman (eds.},
1993.
Academic Press
"RNA-Protein Interactions", Nagai and Mattaj (eds.), 1994. Oxford University
Press
"mRNA Metabolism and Post-transcriptional Gene Regulation", Harford and Morris
(eds.), 1997. Wiley-Liss
Translation
"Translational Control", J.W.B. Hershey, M.B. Mathews, and N. Sonenberg
(eds.), 1995.
Cold Spring Harbor Laboratory Press
General Physiology
"Textbook of Medical Physiology" 9'" Edtion by A.C. Guyton and J.E. Hall W.B.
3o Saunders, 1997
"Review of Medical Physiology", 18'" Edition by W.F. Ganong, Appleton and
Gauge, 1997
Online Databases
Those skilled in the art are familiar with how to search the literature, such
as, e.g.,
libraries, online pubmed, abstract listings, and online mutation databases.
One
particularly useful resource is maintained at the web site of the National
Center for
Biotechnology Information (ncbi): http://www.ncbi.nlm.nih.gov/. From the ncbi
site one
CA 02335649 2001-O1-19
52
WO 00/04194 PCTIUS99/16440
can access Online Mendelian Inheritance in Man (OMIM),. OMIM can be found at:
httw//www3 ncbi nlm nih Qov/Omim/searcllomim html. OMIM is a medically
oriented
database of genetic information with entries for thousands of genes. The OMIM
record
number is provided for many of the genes in Table 10 (see column 3), and
constitutes an
excellent entry point for identification of references that point to the
broader literature.
Another useful site at NCBI is the Entrez browser, located at
1~17~://www3.ncbi.nlm.nihaeov/Entrezl. One can search genomes,
polynucleotides,
proteins, 3D structures, taxonomy or the biomedical literature (PubMed) via
the Entrez
site. More generally links to a number of useful sites with biomedical or
genetic data are
maintained at sites such as Med Web at the Emory University Health Sciences
Center
Library: h~~://WWW.MedWeb.Emoy.Edu/MedWeb/: Riken, a Japanese web site at:
h~tn://www.rtc.riken.go ip/othersite.html with links to DNA sequence,
structural,
molecular biology, bioinformatics, and other databases; at the Oak Ridge
National
Laboratory web site: h~tp:/iwww.ornl.gov/hg_mis/links.html: or at the Yahoo
website of
Diseases and Conditions:
'// ir.vahQo.com/health/diseases and conditions/index.html. Each of the
indicated
web sites has additional useful links to other sites.
Another type of database with utility in selecting the genes on a biochemical
pathway that may affect the response to a drug are databases that provide
information on
biochemical pathways. Examples of such databases include the Kyoto
Encyclopedia of
Genes and Genomes (KEGG), which can be found at:
h~://www.genome.a~,jn_ /keeg,~kegg html. This site has pictures of many
biochemical
pathways, as well as links to other metabolic databases such as the well known
Boehringer Mannheim biochemical pathways charts: h~://www.expas~~ ch/cg.,~
bin/search-biochem-index. The metabolic charts at the latter site are
comprehensive, and
excellent starting points for working out the salient enzymes on any given
pathway.
Each of the web sites mentioned above has links to other useful web sites,
which
in turn can lead to additional sites with useful information.
Research Libraries
Those skilled in the art will often require information found only at large
libraries.
The National Library of Medicine (ht :/~www.nim.nih.gov~ is the largest
medical
library in the world and its catalogs can be searched online. Other libraries,
such as
university or medical school libraries are also useful to conduct searches.
Biomedical
3s books such as those referred to above can often be obtained from online
bookstores as
described above.
Biomedical Literature
CA 02335649 2001-O1-19
WO 00/04194 53 PCTNS99/16440.
To obtain up to date information on drugs and their mechanism of action and
biotransformation; disease pathophysiology; biochemical pathways relevant to
drug
action and disease pathophysiology; and genes that encode proteins relevant to
drug
action and disease one skilled in the art will consult the biomedical
literature . A widely
used, publically accessible web site for searching published journal articles
is PubMed
(h~tp~//www ncbi nlm.nih.gov/PubMedn. At this site, one can search for the
most recent
articles {within the last 1-2 months) or for specific details on methods that
are less recent
(back to 1966). Many Journals also have their own sites on the world wide web
and can
be searched online. For example see the IDEAL web site at:
hlxw//www ap~n~et.com/www/an/aboutid.html. This site is an online library,
featuring full
text journals from Academic Press and selected journals from W.B. Saunders and
Churchill Livingstone. The site provides access (for a fee) to nearly 2000
scientific,
technical, and medical journals.
Experimental methods for identif cation of genes involved in the action of a
drug
There are a number of experimental methods for identifying genes and gene
products that mediate or modulate the effects of a drug or other treatment.
They
encompass analyses of RNA and protein expression as well as methods for
detecting
protein - protein interactions and protein - ligand interactions. Two
preferred
2o experimental methods for identification of genes that may be involved in
the action of a
drug are (1) methods for measuring the expression levels of many mRNA
transcripts in
cells or organisms treated with the drug (2) methods for measuring the
expression levels
of many proteins in cells or organisms treated with the drug.
RNA transcripts or proteins that are substantially increased or decreased in
drug
2s treated cells or tissues relative to control cells or tissues are
candidates for mediating the
action of the drug. Other useful experimental methods include protein
interaction
methods such as the yeast two hybrid system and variants thcreof which
facilitate the
detection of protein - protein interactions.
The pool of RNAs expressed in a cell is sometimes referred to as the
3o transcriptome. Methods for measuring the transcriptome, or some part of it,
are known in
the art. A recent collection of articles summarizing some current methods
appeared as a
supplement to the journal Nature Genetics. (The Chipping Forecast. Nature
Genetics
supplement, volume 21, January 1999.) Experiments have been described in model
systems that demonstrate the utility of measuring changes in the transcriptome
before
35 before and after changing the growth conditions of cells, for example by
changing the
nutritional status. The changes in gene expression help reveal the network of
genes that
mediate physiological responses to the altered growth condition. Similarly,
the addition
of a drug to the cellular or in vivo environment, followed by monitoring the
changes in
CA 02335649 2001-O1-19
PCT/US99/16440.
WO 00/04194 54
gene expression can aid in identification of pharmacological $ene networks.
The pool of proteins expressed in a cell is sometimes referred to as the
proteome.
Studies of the proteome may include not only protein abundance but also
protein
subcellular localization and protein-protein interaction. Methods for
measuring the
proteome, or some part of it, are known in the art. One widely used method is
to extract
total cellular protein and separate it in two dimensions, for example first by
size and then
by isoelectric point. The resulting protein spots can be stained and
quantitated, and
individual spots can be excised and analyzed by mass spectrometry to provide
definitive
identification. The results can be compared from two or more cell lines or
tissues, at least
1o one of which has been treated with a drug. The differential up or down
modulation of
specific proteins in response to drug treatment may indicate their role in
mediating the
pharmacologic actions of the drug. Another way to identify the network of
proteins that
mediate the actions of a drug is to exploit methods for identifying
interacting proteins.
By starting with a protein known to be involved in the action of a drug - for
example the
~ 5 drug target - one can use systems such as the yeast two hybrid system and
variants
thereof (known to those skilled in the art) to identify additional proteins in
the network of
proteins that mediate drug action. The genes encoding such proteins would be
useful for
screening for DNA sequence variances, which in turn may be useful for analysis
of
interpatient variation in response to treatments. For example, the protein 5-
lipoxygenase
20 (SLO) s an enzyme which is a the beginning of the leukotriene biosynthetic
pathway and
is a target for anti-inflammatory drugs used to treat asthma and other
diseases. In order to
detect proteins that interact with 5-lipoxygenase the two-hybrid system was
recently used
to isolate three different proteins, none previously known to interact with
SLO. (Provost
et al., Interaction of 5-lipoxygenase with cellular proteins. Proc. Natl.
Acad. Sci. U.S.A.
25 96: 1881-1885, 1999.) A recent collection of articles summarizing some
current methods
in proteomics appeared in the August 1998 issue of the journal Electrophoresis
(volume
19, number 11). Other useful articles include: Blackstock WP, et al.
Proteomics:
quantitative and physical mapping of cellular proteins. Trends Biotechnol. 17
(3): p. 121-
7, 1999, and Patron W.F., Proteome analysis II. Protein subcellular
redistribution: linking
3o physiology to genomics via the proteome and separation technologies
involved. J
Chromatogr B Biomed Sci App. 722(1-2):203-23. 1999.
Since many of these methods can also be used to assess whether specific
polymorphisms are likely to have biological effects, they should also be
considered as
relevant in section 3, below, concerning methods for assessing the likely
contribution of
35 variances in candidate genes to clinical variation in patient responses to
therapy.
2. Screen for Variances in Genes that may be Related to Therapeutic Response
Having identified a set of genes that may affect response to a drug the next
step is
to screen the genes for variances that may account for interindividual
variation in
CA 02335649 2001-O1-19
WO 00/04194 PCTNS99/16440
response to the drug. There are a variety of levels at which a gene can be
screened for
variances, and a variety of methods for variance screening. The two main
levels of
variance screening are genomic DNA screening and cDNA screening. Genomic
variance
detection may include screening the entire genomic segment spanning the gene
from the
5 transcription start site to the polyadenylation site. Altennatively genomic
variance
detection may (for intron containing genes) include the exons and some region
amend
them containing the splicing signals, for example, but not all of the intronic
sequences.
In addition to screening intmns and exons for variances it is generally
desirable to screen
regulatory DNA sequences for variances. Promoter, enhancer, silencer and other
1o regulatory elements have been described in human genes. The promoter is
generally
proximal to the transcription start site, although there may be several
promoters and
several transcription start sites. Enhancer, silencer and other regulatory
elements may be
intragenic or may lie outside the introns and exons, possibly at a
considerable distance,
such as 100 kb away. Variances in such sequences may affect basal gene
expression or
15 regulation of gene expression. In either case such variation may affect the
response of an
individual patient to a therapeutic intervention, for example a drug, as
described in the
examples. Thus in practicing the present invention it is useful to screen
regulatory
sequences as well as transcribed sequences, in order to identify variances
that may affect
gene transcription. Frequently information on the genomic sequence of a gene
can be
2o found in the sources above, particularly by searching GenBank or Medline
(PubMed).
The name of the gene can be entered at a site such as Entrez:
h~~p~//www ncbi nlm nih govBntrez/nucleotide html. Using the genomic sequence
and
information from the biomedical literature one skilled in the art can perform
a variance
detection procedure such as those described in examples 14, 15 and 16.
2s Variance detection is often first performed on the cDNA of a gene for
several
reasons. First, available data on functional sequence variances suggests that
variances in
the transcribed portion of a gene are most likely to have functional
consequences as they
can affect the interaction of the transcript with a wide variety of cellular
factors during the
complex processes of transcription, processing and translation. Second, as a
practical
3o matter the cDNA sequence of a gene is often available before the genomic
structure is
known, although the reverse may be true in the future as the sequence of the
human
genome is determined. If the genomic structure is not known then only the cDNA
seqence can be scanned for variances. Methods for preparing cDNA are described
in
Example 13. Methods for variance detection on cDNA are described below and in
the
35 examples.
Methods for variance screening have been described, including DNA sequencing.
See for example: US5698400: Detection of mutation by resolvase cleavage;
US5217863:
Detection of mutations in nucleic acids; and US5750335: Screening for genetic
variation,
CA 02335649 2001-O1-19
56
WO 00/04194 PCTIUS99/1I440
as well as the examples and references cited therein for examples of useful
variance
detection procedures. Detailed variance detection procedures are also
described in
examples 14, 15 and 16. One skilled in the art will recognize that depending
on the
specific aims of a variance detection project (number of genes being screened,
number of
individuals being screened, total length of DNA being screened) one of the
above cited
methods may be preferable to the others, or yet another procedure may be
optimal. A
preferred method of variance detection is chain terminating DNA sequencing
using dye
labeled primers, cycle sequencing and software for assessing the quality of
the DNA
sequence as well as specialized software for calling heterozygotes. The use of
such
t0 procedures has been described by Nickerson and colleagues. See for example:
Rieder
M.J., et al. Automating the identification of DNA variations using quality-
based
fluorescence re-sequencing: analysis of the human mitochondria) genome.
Nucleic Acids
Res. 26 (4):967-73,1998, and: Nickerson D.A., et al. PolyPhred: automating the
detection
and genotyping of single nucleotide substitutions using fluorescence-based
resequencing.
Nucleic Acids Res. 25 (14):2745-51,1997.Although the variances provided in
tables 3, 4,
and 10 consist principally of cDNA variances, it is a part of this invention
that detection
of genomic variances is also a useful method for identification of variances
that may
account for interpatient variation in response to a therapy.
3. Assess the Likely Contribution of Variances in Candidate Genes to Clinical
Variation in Patient Responses to Therapy
Once a set of genes likely to affect disease pathophysiology or drug action
has
been identified, and those genes have been screened for variances, said
variances (e.g.,
provided in Tables 3, 4, and 10) can be assessed for their contribution to
variation in the
pharmacological or toxicological phenotypes of interest. There are several
methods
which can be used in the present invention for assessing the medical and
pharmaceutical
implications of a DNA sequence variance. They range from computational methods
to in
vitro and/or in vivo experimental methods (discussed below), to prospective
human
clinical trials (see below), and also include a variety of other laboratory
and clinical
measures that can provide evidence of the medical consequences of a variance.
In
general, human clinical trials constitute the highest standard of proof that a
variance or set
of variances is useful for selecting a method of treatment, however,
computational and in
vitro data, or retrospective analysis of human clinical data may provide
strong evidence
that a particular variance will affect response to a given therapy. Moreover,
at an early
stage in the analysis when there are many possible hypotheses to explain
interpatient
variation in treatment response, the use of informatics-based approaches to
evaluate the
likely functional effects of specific variances is an efficient way to
proceed.
CA 02335649 2001-O1-19
57
WO 00/04194 PCTNS99I16440
Informatics-based approaches to the prediction of the.likely functional
effects of
variances include DNA and protein sequence analysis (phylogenetic approaches
and
motif searching) and protein modeling (based on coordinates in the protein
database, or
pdb; see http://www.rcsb.org/pdbn. Such analyses can be performed quickly and
inexpensively, and the results allow selection of certain genes for more
extensive in vitro
or in vivo studies (see below) or for more variance detection (see above) or
both.
More specifically, the structure of many medically and pharmaceutically
important proteins, or homologs of such proteins in other species, or examples
of
domains present in such proteins, is known. Further, there are increasingly
powerful
tools for modeling the structure of proteins with unsolved structure,
particularly if there is
a related (e.g., a homologous) protein with known structure. (For reviews see:
Rost et al.,
Protein fold recognition by prediction-based threading, J. Mol. Biol. 270:471-
480, 1997;
Firestine et al., Threading your way to protein fimction, Chem. Biol. 3:779-
783, 1996)
There are also powerful methods for identifying conserved domains and vital
amino acid
residues of proteins of unknown structure by analysis of phylogenetic
relationships.
(Deleage et al., Protein structure prediction: implications for the biologist,
Biochimie
79:681-686, 1997; Taylor et al., Multiple protein structure alignment, Protein
Sci.
3:1858-1870, 1994) These methods can permit the prediction of functionally
important
variances, either on the basis of structure or evolutionary conservation. For
example, a
crystal structure can reveal which amino acids comprise a small molecule
binding site.
The identification of a polymorphic amino acid variance in the topological
neighborhood
of such a site, and in particular, the demonstration that at least one variant
form of the
protein has a variant amino acid which impinges on the known small molecule
binding
pocket differently from another variant form, provides strong evidence that
the variance
affects the function of the protein. From this it follows that the interaction
of the protein
with a treatment method, such an administered drug, will also likely be
altered. One
skilled in the art will recognize that the application of computational tools
to the
identification of functionally consequential variances involves applying the
knowledge
and tools of medicinal chemistry and physiology to the analysis.
Phylogenetic approaches to understanding sequence variation are also useful.
Thus if a sequence variance occurs at a nucleotide or encoded amino acid
residue where
there is usually little or no variation in homologs of the protein of interest
from non-
human species, particularly evolutionarily remote species, then the variance
is more
likely to affect function of the RNA or protein.
4. Perform in vitro or in vivo Experiments to Assess the Functional Importance
of
Gene Variances
CA 02335649 2001-O1-19
58
WO 00/04194 PCT/US99/16440
The selection of an appropriate experimental program.for testing the medical
consequences of a variance may differ depending on the nature of the variance,
the gene,
and the disease. For example if there is already evidence that a protein is
involved in the
pharmacologic action of a drug, then the in vitro demonstration that an amino
acid
variance in the protein affects its biochemical activity is strong evidence
that the variance
will have an effect on the pharmacology of the drug in patients, and therefore
that patients
with different variant forms of the gene may have different responses to the
same dose of
drug. If the variance is silent with respect to protein coding information, or
if it lies in a
noncoding portion of the gene (e.g., a promoter, an intron, or a 5'- or 3'-
untranslated
1o region) then the appropriate biochemical assay may be to assess mRNA
abundance, half
life, or translational efficiency. If, on the other hand, there is no
substantial evidence that
the protein encoded by a particular gene is relevant to drug pharmacology,
then the
appropriate test is a clinical study addressing the responses to therapy of
two patient
groups distinguished on the basis of one or more variances. This approach
reflects the
current reality that biologists do not sufficiently understand gene regulation
and gene
expression to consistently make accurate inferences about the consequences of
DNA
sequence variances.
Thus, if there is a reasonable hypothesis regarding the effect of a protein on
the
action of a drug, then the in vitro and in vivo approaches described below
will usefully
2o predict whether a given variance is therapeutically consequential. If, on
the other hand,
there is no evidence of such an effect, then the most appropriate test is the
empirical
clinical measure of efficacy (which requires no evidence or assumptions
regarding the
mechanism by which the variance may exert an effect on a therapy). Clinical
studies may
be performed either prospectively or retrospectively.
Experimental Methods: Genomic DNA Analysis
Variances in DNA may affect the basal transcription or regulated transcription
of
a gene locus. Such variances may be located in any part of the gene but are
most likely to
be located in the promoter region, the first intron, or in 5' or 3' flanking
DNA, where
3o enhancer or silencer elements may be located. Methods for analyzing
transcription are
well known to those skilled in the art and exemplary methods are described in
some of
the texts cited below. Transcriptional run off assay is one useful method.
Detailed
protocols for useful methods can be found in texts such as: S~rrent Protocols
in
MnlPenla_r Bi_eloev edited by: F.M. Ausubel, R.Brent, R.E. Kingston, D.D.
Moore, J.G.
Seidman, K. Struhl, John Wiley & Sons, Inc, 1999, or: Molecular Cloning: A
Laboratory
Manual by J. Sambrook, E.F. Fritsch and T Maniatis. 1989. 3 vols, 2nd edition,
Cold
Spring Harbor Laboratory Press
CA 02335649 2001-O1-19
59
PCT/US99/16440
WO 00/04194
Experimental Methods: RNA Analysis
RNA variances may affect a wide range of processes including RNA splicing,
polyadenylation, capping, export from the nucleus, interaction with
translation intiation,
elongation or termination factors, or the ribosome, or interaction with
cellular factors
including regulatory proteins, or factors that may affect mRNA half life.
However, any
effect of variances on RNA function should ultimately be measurable as an
effect on
RNA levels - either basal levels or regulated levels or levels in some
abnormal cell state.
Therefore one preferred method for assessing the effect of RNA variances on
RNA
function is to measure the levels of RNA produced by different alleles in one
or more
1o conditions of cell or tissue growth. Said measuring can be done by
conventional methods
such as Northern blots or RNAase protection assays (kits available from
Ambion, Inc.),
or by methods such as the Taqman assay (developed by the Applied Biosystems
Division
of the Perkiz~ Elmer Corporation), or by using arrays of oligonucleotides or
arrays of
cDNAs attached to solid surfaces. Systems for arraying cDNAs are available
commercially from companies such as Nanogen and General Scanning. Complete
systems for gene expression analysis are available from companies such as
Molecular
Dynamics. For recent reviews of the technology see the supplement to volume 21
of
Nature Genetics entitled "The Chipping Forecast", especially articles
beginning on pages
9, 15, 20 and 25.
2o Additional methods for analyzing the effect of variances on RNA include
secondary structure probing, and direct measurement of half life or turnover.
Secondary
structure can be determined by techniques such as enzymatic probing (using
enzymes
such as T1, T2 and S1 nuclease), chemical probing or RNAase H probing using
oligonucleotides. Some RNA structural assays can be performed in vitro or on
cell
extracts or on
Experimental Methods: Protein Analysis
There are a variety of experimental methods for investigating the effect of a
variance on response of a patient to a treatment. The preferred method will
depend on the
availability of cells expressing a particular protein, and the feasibility of
a cell-based
assay vs. assays on cell extracts, on proteins produced in a foreign host, or
on proteins
prepared by in vitro translation.
For example, the methods and systems listed below can be utilized to
demonstrate
differential expression and/or activity, or in model system phenotype/genotype
correlations.
For the determination of protein levels or protein activity one could utilize
a
variety of techniques. The in vitro protein activity can be determined by
transcription or
translation in bacteria, yeast, baculovirus, COS cells (transient), CHO, or
study directly in
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
human cells: Further, one could perform pulse chase for experiments for the
determination of changes in protein stability (half life).
One skilled in the art could manipulate the cell assay to address grouping the
cells
by genotypes or phenotypes. For example, identification of cells with
different genotypes
s (possibly including families) and phenotype may be performed using
standardized
laboratory molecular biological protocols. After identification and grouping,
one skilled
in the art could determine whether there exists a correlation between cellular
genotype
and cellular phenotype.
Advancing an experimental preclinical program may include testing these in
vitro
1o hypotheses in vivo, e.g. an animal model. For example, one skilled in the
art would
readily have the ability to create gene knockouts. In this case, an embryonic
stem cell is
genetically manipulated to be deficient in a given gene. More specifically, a
DNA
construct is created that will undergo homologous recombination when inserted
into the
said embryonic stem cell nucleus. After the recombination event has occurred,
the
15 targeted gene is effectively inactivated due to the insertion of sequence
(usually a
translation stop or a marker gene sequence). This can be accomplished in
worms,
drosophila, or mice. The species chosen will be conducive to attain maximal
experimental results for the particular gene and the particular variance,
variances, or
haplotype. Once the knockout species is created the candidate therapeutic
intervention
2o can be administered to the animal and tested for effects on gene expression
or effects of
various gene deficiencies. In the case whereby the chosen cell is a lower
eukaryote, e.g.
yeast, genetic manipulation occurs via introduction of a DNA construct that
will undergo
homologous recombination to disrupt the endogenous gene or genes.
The methods described above are reviewed and compiled in the following list of
2s texts.
General Molecular Bi~IoQV Methods
"Molecular Biology: A project approach", S.J. Karcher, Fall 1995. Academic
Press
30 "DNA Cloning: A Practical Approach", D.M. Glover and B.D. Hayes (eds).
1995.
IRL/Oxford University Press. Vol. 1 - Core Techniques; Vol 2 - Expression
Systems;
Vol. 3 - Complex Genomes; Vol. 4 -Mammalian Systems.
"Short Protocols in Molecular Biology", Ausubel et al. October 1995. 3rd
edition,
John Wiley and Sons
35 Current Protocols in Molecular Biology Edited by: F.M. Ausubel, R.Brent,
R.E.
Kingston, D.D. Moore, J.G. Seidman, K. Struhl, (Series Editior: V.B. Chanda),
1988
"Molecular Cloning: A laboratory manual", J. Sambrook, E.F. Fritsch. 1989. 3
vols,
2nd edition, Cold Spring Harbor Laboratory Press
CA 02335649 2001-O1-19
61
WO 00/04194 PCT/US99/16440
Polymerase chain reaction (PCR)
"PCR Primer: A laboratory manual", C.W. Diffenbach and G.S. Dveksler (eds.),
1995. Cold Spring Harbor Laboratory Press
"The Polymerase Chain Reaction", K.B. Mullis et al. (eds.), 1994. Birkhauser
"PCR Strategies", M.A. Innis, D.H. Gelf, and J.1. Sninsky (eds.), 1995.
Academic
Press
General procedures for discipline specific studies
to Current Protocols in Neuroscience Edited by: J. Crawley, C. Gerfen, R.
McKay,
M. Rogawski, D. Sibley, P. Skolnick, (Series Editor: G. Taylor), 1997
Current Protocols in Pharmacology Edited by: S. J. Enna / M. Williams, J.W.
Ferkany, T. Kenakin, R.E. Porsolt, J.P. Sullivan, (Series Editor: G.
Taylor),1998
Current Protocols in Protein Science Edited by: J.E. Coligan, B.M. Dunn, H.L.
15 Ploegh, D.W. Speicher, P.T. Wingfield, (Series Editor: Virginia Benson
Chanda), 1995
Current Protocols in Cell Biology Edited by: J.S. Bonifacino, M. Dasso, J.
Lippincott-Schwartz, J.B. Harford, K.M. Yamada, (Series Editor: K. Morgan)
1999
Current Protocols in Cytometry Managing Editor: J.P. Robinson, Z.
Darzynkiewicz (ed) / P. Dean (ed), A. Orfao (ed), P. Rabinovitch (ed), C.
Stewart (ed), H.
2o Tanke (ed), L. Wheeless (ed), (Series Editor: J. Paul Robinson), 1997
Current Protocols in Human Genetics Edited by: N.C. Dracopoli, J.L. Haines,
B.R. Korf, D.T. Moir, C.C. Morton, C.E. Seidman, J.G. Seidman, D.R. Smith,
(Series
Editor: A. Boyle), 1994
Current Protocols in Immunology Edited by: J.E. Coligan, A.M. Kruisbeek, D.H.
25 Margulies, E.M. Shevach, W. Strober, (Series Editor: R. Coico), 1991
III. Clinical Trials
A clinical trial is the definitive test of the utility of a variance or
variances for the
3o selection of optimal therapy. Clinical trials require no knowledge of the
biological
function of the gene containing the variance or variances to be assessed, nor
any
knowledge of how the therapeutic intervention to be assessed works at a
biochemical
level; the question of the utility of a variance can be addressed at a purely
phenomenological level. On the other hand, if there is information about
either the
35 biochemical basis of a therapeutic intervention or the biochemical effects
of a variance,
then a clinical trial can be designed to test a specific hypothesis.
Methods for performing clinical trials are well known in the art. ( 'Guide to
clinical Trials by Bert Spilker, Raven Press, 1991; The Ranc~Om»ed Clinical
Trial and
CA 02335649 2001-O1-19
62
WO 00/04194 PCT/US99/16440
~~neutic Decisions by Niels Tygstrup (Editor), Marcei Dekker; Recent Advances
in
Clinical Trial Design and Analysis {Cancer Treatment and Research, Ctar 75) by
Peter F.
Thall (Editor) Kluwer Academic Pub, 1995. However, performing a clinical trial
to test
the genetic contribution to interpatient variation in drug response requires
some
additional design considerations, including defining what the genetic
hypothesis is, how
it is to be tested, how many patients will need to be enrolled to have
adequate statistical
power to measure an effect of a specified magnitude (power analysis),
definition of
primary and secondary endpoints, and methods of statistical analysis, as well
as other
aspects. In the outline below some of the major types of genetic hypothesis
testing,
~ o power analysis, statistical analysis, etc. are summarized. One skilled in
the art will
recognize that certain of the methods will be best suited to specific clinical
situations, and
that additional methods are known and can be used in particular instances.
A. Performing a Clinical Trial
~5 As used herein, a "clinical trial" is the testing of a therapeutic
intervention in a
volunteer human population for the purpose of determining whether a
therapeutic
intervention is safe and/or efficacious in the human volunteer or patient
population for a
given disease, disorder, or condition. The analysis of safety and efficacy in
genetically
defined subgroups differing by at least one variance is of particular
interest.
2o A "clinical study" is that part of a clinical trial that involves
determination of the
effect a candidate therapeutic intervention on human subjects. It includes
clinical
evaluations of physiologic responses including pharmacokinetic (absorption,
distribution,
bioavailability, and excretion) as well as pharmacodynamic (physiologic
response and
efficacy) parameters. A pharmacogenetic clinical study is a clinical study
that involves
25 testing of one or more specific hypotheses regarding the effect of a
genetic variance or
variances (or set of variances, i.e. haplotype or haplotypes) in enrolled
subjects or patients
on response to a therapeutic intervention. These hypotheses are articulated
before the
study in the form of primary or secondary endpoints. For example the endpoint
may be
that in a particular genetic subgroup the rate of objectively defined
responses exceeds
3o some predefined threshold.
For each clinical study to commence enrollment and proceed to treat subjects
at a
given institution, an application that describes in detail the scientific
premise for the
therapeutic intervention and the procedures involved in the study, including
the endpoints
and analytical methods to be used in evaluating the data must be reviewed and
accepted
35 by regulatory authorities at the level of the institution and the federal
government (in the
U.S.). In the U.S., there are two regulatory bodies that oversee conduct of
clinical trials:
an Institutional Review Board (IRB) and the United States Food and Drug
Administration
(US FDA). The European counterpart of the US FDA is the European Medicines
CA 02335649 2001-O1-19
63
WO 00/04194 PCT/US99/16440
Evaluation Agency (EMEA). Similar agencies exist in other countries.
An Institutional Review Board accepts and reviews applications for clinical
trials
that are to be conducted at the institution and are to include healthy
volunteers or human
subjects from a defined patient population that seeks medical, surgical,
rehabilitative, or
social services at that institution. The application includes document
sections that
provide the rationale for and describe the scope of the clinical study. For
example, an
application to an IRH may include a clinical protocol, and informed consent
forms.
It is also customary, but not required, to prepare an investigator's brochure
which
describes the scientific hypothesis for the proposed therapeutic intervention,
the
to preclinical data, and the clinical protocol in concise language. The
brochure is made
available to any physician participating in the proposed or ongoing trial. The
investigator's brochure for a phannacogenetic clinical trial will include a
full description
of the genetic variance and/or variances believed or hypothesized to account
for
differential responses in the normal human subjects or patients; as well as a
description of
the genetic statistical analysis.
The supporting preclinical data is a report of all the in vitro, in vivo
animal or
previous human trial data that supports the safety and/or efficacy of a given
therapeutic
intervention. In a pharmacogenetic clinical trial the preclinical data may
also include a
description of the effect of a specific genetic variance or variances on
biochemical or
physioiogic experimental variables, or on treatment outcomes, as determined by
in vitro
studies or by retrospective genetic analysis of clinical trial or other
medical data (see
below) used to first formulate or test a pharmacogenetic hypothesis.
The clinical protocol provides the relevant scientific and therapeutic
introductory
information, describes the inclusion and exclusion criteria for human subject
enrollment,
including genetic criteria if relevant, describes in detail the exact
procedure or procedures
for treatment using the candidate therapeutic intervention, describes
laboratory analyses
to be performed during the study period, and lastly describes the risks (both
known and
unknown) involving the use of the experimental candidate therapeutic
intervention. In a
clinical protocol for a pharmacogenetic clinical trial, the clinical protocol
will further
3o describe the gene or genes believed or hypothesized to affect differential
patient
responses and the variance or variances to be tested. Further, the clinical
protocol for a
pharmacogenetic clinical trial will include a description of the
stratification of the
treatment groups based on one or more gene sequence variances or combination
of
variances or haplotypes.
The informed consent document is a description of the therapeutic intervention
and the clinical protocol in simple language (third grade level) for the
patient to read,
understand, and, if willing, agree to participate in the study by signing the
document. In a
pharmacogenetic clinical study the informed consent document will describe, in
simple
CA 02335649 2001-O1-19
64
WO 00/04194 PCT/US99/16440
language, the use of a genetic test or a limited set of genetic tests to
determine the subject
or patients status at a particular gene variance or variances, and to further
ascertain
whether, in the study population, particular variances are associated with
particular
clinical or physiological responses.
The US FDA reviews proposed clinical trials through the process of an
Investigational New Drug Application (IND). The IND is composed of the
investigator's
brochure, the supporting in vitro and in vivo animal or previous human data,
the clinical
protocol, and the informed consent documents or forms. In each of the sections
of the
IND, a specific description of a single allelic variance or a number of
variances to be
1o tested in the clinical study will be included. For example, in the
investigator's brochure a
description of the gene or genes believed or hypothesized to account, at least
in part, for
differential responses will be included as well as a description of genetic
variance or
variances of a particular candidate gene or genes. Further, the preclinical
data may
include a description of in vivo or in vitro studies of the biochemical or
physiologic
t 5 effects of a variance or variances (e.g., haplotype) in a candidate gene
or genes, as well as
the predicted effects of the variance or variances on efficacy or toxicology
of the
candidate therapeutic intervention. Alternatively the results of retrospective
genetic
analysis of response data in patients treated with the candidate therapy may
be the basis
for formulating the genetic hypotheses to be tested in the prospective trial.
For first in
2o man clinical studies, the focus of this section will be safety. The US FDA
reviews the
application with a particular emphasis on the safety data and whether
toxicological data is
supportive and sufficient to justify proceeding to human testing.
The established phases of clinical development are Phase I, II, III, and IV.
The
fundamental objectives for each phase become increasingly complex as the
stages of
2s clinical development progress. In Phase I, safety in humans is the primary
focus. In
these studies, dose-ranging designs establish whether the candidate
therapeutic
intervention is safe in the suspected therapeutic concentration range. In a
pharmacogenetic clinical trial there may be an analysis of the effect of a
variance or
variances on Phase I safety or surrogate efficacy parameters. At the same
time,
3o pharmacokinetic parameters (e.g., adsorption, distribution, metabolism, and
excretion)
may be a secondary objective. In a pharmacogenetic clinical study, there may
be
additional analysis of the gene or genes and allelic variance or variances
that are
suspected to be involved in these pharmacokinetic parameters. As clinical
development
stages progress, trial objectives focus on the appropriate dose to elicit a
therapeutically
35 relevant response. In a pharmacogenetic clinical trial, the dose or doses
selected may be
different than those identified based upon preclinical safety and efficacy
determinations.
For example, phenotypic effects of an allele depends on its frequency and also
its
interaction with the environment, as described earlier. Therefore, once the
frequency of
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
an allele or haplotype has been established for selected human subjects or
patients, the
effect of the variance on the drug responses by perfonming both in vitro or in
vivo
analyses under controlled conditions. Under these conditions, drug dosage
could be
adjusted accordingly. In some instances, the chosen dose may be one that is
sub-optimal
5 or is significantly less toxic so that determination of the effect of
allelic variance or
variances for a given treatment or human volunteer population may be
appropriately
tested and analyzed. In other instances, the dose may be similar to or the
same as that
chosen based upon in vitro or in vivo data. In yet other instances, the dose
may be greater
than optimal because allelic differences or haplotypes may result in enhanced
elimination,
10 metabolic inactivation, or excretion.
Lastly, the objectives in the latter stages of clinical development center on
the
effect of the therapeutic intervention on the general population. In these
trials, the
numbers of individuals required for enrollment and the number of treatment
conditions
required to achieve the objectives of the trial is dictated by statistical
power analysis. The
15 number of patients required for a given pharmacogenetic clinical trial will
be determined
on the prior knowledge of but not exclusively limited to variance or haplotype
frequency,
actual disease, disorder, or condition causing allele or allele associated
with the disease,
disorder, or condition and their linkage relationships. For a large scale
pharmacogenetic
clinical study, the identified sample size will require an adequate analysis
of the
2o frequency of the allelic variance or variances within a given population,
as described, for
example, by Tu & Whitkemore (1999) and references therein.
Clinical trials can be designed to obscure the human subjects and/or the study
coordinators from biasing that may occur during the testing of a candidate
therapeutic
invention. Often the candidate therapeutic intervention is compared to best
medical
25 treatment, or a placebo (a compound, agent, device, or procedure that
appears identical to
the candidate therapeutic intervention but is innocuous to the receiving
subject). Thus,
control with placebo limits efficacy perception by influencing factors such as
prejudice
on the part of the study participant or investigator, spontaneous alterations
or variations
that occur during treatment and are related to the disease studied, or are
unrelated to the
3o candidate therapeutic intervention. In pharmacogenetic clinical studies, a
placebo arm or
best medical therapy may be required in order to ascertain the effect of the
allelic
variance or variances on the efficacy or toxicology of the candidate
therapeutic
intervention.
Blinding refers to the lack of knowledge of the identity of the trial
treatment and
35 thus can be used to ascertain the real and not perceived effects of the
candidate
therapeutic intervention. Patients, trial subjects, investigators, data review
committees,
ancillary personnel, statisticians, and clinical trial monitors may be blinded
or unblinded
during the trial period. Open label trials refer to those that are unblinded;
single blind is
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/1b440
when the patient is kept unaware of the treatment groups; double blind is when
both the
patient and the investigator is kept unaware of the treatment groups; or a
combination of
these may be instituted during the trial period. PharTnacogenetic clinical
trial design may
include one or a combination of open label, single blind, or double blind
clinical trial
design because reduction of inherent biases due to the knowledge of the type
of
treatment the human subject or the patient is to receive will enswe detection
of the
accwacy of the benefits of the stratification based upon allelic variance or
variances or
haplotypes.
In the designed studies in all fow phases, termination endpoints for trials
1o including or excluding pharmacogenetic objectives are defined and include
observation of
adverse clinical events, voluntary lack of study participation either in the
form of lack of
adherence to the clinical protocol or sudden change in lifestyle of the
participant, lack of
adherence on the part of trial investigators to follow the trial protocol,
death, or lack of
efficacy or positive response within the test group.
Phase I of clinical development is a safety study performed in a limited (<
15)
number of normal, healthy volunteers usually at single institutions. The
primary
endpoints in these studies is to determine pharmacokinetic parameters (i.e.
adsorption,
distribution, and bioavailability), dose-related side effects that are either
desirable or
undesirable, and metabolites that corroborate preclinical animal studies. In a
Phase I
2o pharmacogenetic clinical trial, stratification based upon allelic variance
or variances of a
suspected gene or genes involving any or all of the pharmacokinetic parameters
will be
considered and incorporated in the objectives of the trial design.
In some cases, a pharmacogenetic Phase I study may enroll healthy human
volunteers and stratify these individuals based upon their genotype. In this
case, a study
objective may include observation of the effect of the allele/haplotype
(detectable or
undetectable) which the candidate therapeutic intervention may exhibit within
the allelic
variance, allelic variances, or haplotype groupings which can be assessed in
the absence
of a disease, disorder, or condition.
In some cases (e.g. cancer or medically intractable, life threatening, for
those in
3o which no medical alternative exists, or seriously debilitating diseases,
disorders, or
conditions) Phase I studies can include a limited number of patients with a
diagnosed
disease, disorder, or condition for whom clinical parameters satisfy a
specified inclusion
criteria (see below). These safety/limited efficacy studies can be conducted
at multiple
institutions to ensure enrollment of these patients. In a pharmacogenetic
Phase I study
that will include patients to some degree, the gene or genes and allelic
variance or
variances suspected to be involved in the efficacy of the candidate
therapeutic
intervention will be considered in the design of the inclusion criteria, the
objectives, and
the primary endpoints.
CA 02335649 2001-O1-19
67
WO 00/04194 PCT/US99/16440
Phase II studies include a limited number of patients (<100) that satisfy the
required inclusion criteria and do not satisfy any of the exclusion criteria
of the trial
design. Phase II studies can be conducted at single or multiple institutions.
Inclusion
criteria for patient enrollment to a clinical trial is a list of qualities for
a given patient
population that includes pathophysiologic clinical parameters for a given
disease,
disorder, or condition that can be determined by clinical diagnosis or
laboratory or
diagnostic test; age; gender; fertility state (e.g. pre- or postmenopausal
women);
coexisting medical therapies; or psychological, emotional, or cognitive state.
Inclusion
criteria can also include defined psychological, emotional, or socioeconomic
support by
family or friends. Exclusion criteria for patient enrollment generally
includes the listing
of co-morbidities that may interfere with the observations of the medical or
laboratory
pathophysiological clinical parameters of the disease, disorder, or condition,
age, gender,
fertility state (e.g. pre- or postmenopausal women), or previous or concurrent
medical,
surgical, or diagnostic therapies. In Phase iI, the primary endpoint of the
study is
generally limited efficacy and corroboration ofthe Phase I safety data in the
specified
patient population defined by the inclusion/exclusion criteria of the clinical
protocol.
Primary efficacy endpoints include observed improvements of pathophysiologic
parameters that are determined medically, diagnostically (e.g. clinical
laboratory values),
or by surrogate measurements of the pathological state of the disease,
disorder, or
2o condition. Primary endpoints may also include limitation of pharmacologic
therapies,
reduction of time to death, or reduction in the progression of the disease,
disorder, or
condition. Surrogate markers are pathophysiologic parameters determined by
medical or
clinical laboratory diagnosis that are associated and have been correlated
with the
prognosis, progression, predisposition, or risk analysis with a disease,
disorder, or
condition that are not directly related to the primary diagnosed
pathophysiologic
condition, e.g. lowering blood pressure and coronary heart disease. Secondary
endpoints
are those that supplement the primary endpoint and can be used to support
further clinical
studies. For example, secondary endpoints include reduction in pharmacologic
therapy,
reduction in requirement of a medical device, or alteration of the progression
of the
disease disorder, or condition. Typically, in Phase II, treatment groups with
varying
doses are included in the study to identify the appropriate dosage and
pharmacokinetic
parameters to achieve maximum efficacy.
In a pharmacogenetic Phase II clinical trial, retrospective or prospective
design
will include the stratification of the patients based upon suspected gene or
genes and
allelic variance or variances involved in the pathway for pharmacodynamic or
pharmacokinetic response demonstrated in the treatment groups of the candidate
therapeutic intervention. These pharmacodynamic parameters may include
surrogate
endpoints, efficacy endpoints, or pathophysiologic thresholds. Pharmacokinetic
CA 02335649 2001-O1-19
68
WO 00/04194 PCT/US99/1b440
parameters may include but are not exclusive of dosage, toxicological
variables,
metabolism, or excretion. Other parameters that may effect the outcome of a
pharmacogenetic clinical trial may include gender, race, ethnic origins
(population
history), and combination of allelic variances of genes from multiple
pathways, leading to
but not exclusively efficacy or toxicology.
Phase III studies include mufti-site, large, statistically significant,
numbers of
patients (<5,000) that fulfill the inclusion criteria for the study. The
design of this type of
trial includes power analysis to ensure the data will support the study
objectives. In this
large scale efficacy study, the primary endpoint is preferably defined as
enhanced
t0 efficacy as compared to placebo or best medical care for said disease,
disorder, or
condition. The primary endpoint may include reduction of condition
progression,
improvement of a specific subset of symptoms, or in requirement or perceived
need of
medical therapy. In a pharrnacogenetic Phase III clinical study, the endpoints
will be the
determination of the efficacy or toxicological differences that can be
demonstrated to be
t5 dependent on the stratification based upon allelic variance or variances in
a gene or genes
that are suspected to be involved in the efficacy or toxicological population
phenotype.
Further in the Phase III pharmacogenetic clinical trial, the analysis of the
impact of the
allelic variance or variances will be broadened from the confirmatory Phase II
phanmacogenetic clinical trial data that supports the notion that the
phenotypic response
20 differences can be identified as dependent on the allelic variance or
variances of a gene or
genes suspected to be involved in the efficacy or toxicological response.
After the completion of a Phase III study, the data and information from all
of the
trials are compiled into a New Drug Application for review by the US FDA for
marketing
approval in the US and its territories. The NDA includes the raw (unanalyzed)
clinical
25 data, i.e. the primary endpoints or secondary endpoints, a statistical
analysis of all of the
included data, a document describing in detail any adverse or observed side
effects,
tabulation of the participant drop-outs and detailed reasons for the
termination, and other
specific data or details of ongoing in vitro or in vivo studies since the
submission of the
IND. If pharmacoeconomic objectives are a part of the clinical trial design
data
30 supporting cost or economic analyses are included in the NDA. In a
pharmacogenetic
clinical study, the pharmacoeconomic analyses may include demonstration or
lack of
benefit of the candidate therapeutic intervention in a cost benefit analysis,
cost of illness
study, cost minimization study, or cost utility analysis. In one or a
combination of these
studies, the effect of a diagnostic identification of the population and
subsequent
35 stratification based upon allelic variance or variances or haplotype of a
suspected gene or
genes involved in the efficacy or toxicological responses of the candidate
therapeutic
intervention will be used to support application for the approval for the
marketing and
sale of the candidate therapeutic intervention.
CA 02335649 2001-O1-19
69
WO 00/04194 PCT/US99/16440
Phase N studies occur after the therapeutic intervention has been approved for
marketing. In these studies, retrospective data and data from a large patient
population
that do not necessarily fulfill the pathophysiologic requirements of the
approved
indication are included. In a Phase IV pharmacogenetic clinical trial, both
retrospective
and prospective design can be incorporated. In both cases, stratification
based upon
allelic variance or variances with adequate sample size in order to determine
the statistical
relevance of an outcome difference among the treatment groups.
Although the above listed phases of clinical development are well-established,
there are cases whereby strict Phase I, II, III development does not occur,
i.e. the clinical
1o development of candidate therapeutic interventions for serious debilitating
or life
threatening diseases, or for those cases whereby no medical therapeutic
alternative exists.
In the cases whereby the target indication for cancer or medically
intractable, life
threatening or seriously debilitating diseases, disorders, or conditions the
US FDA has
regulatory procedural mechanisms that can expedite the availability of the
therapeutic
intervention for patients that fall into one or more of these categories. Such
development
incentives include Treatment IND, Fast-Track or Accelerated review, and Qrphan
Drug
Status. In a pharmacogenetic clinical development program for candidate
therapeutic
interventions for this class of indications, consideration of sample size for
adequate
determination of the effect allelic variance or variances may have on the
outcome
2o response or endpoints is incorporated. Further consideration may include
but is not
limited to accrual rate for candidate patients, and number of institutions or
clinical sites
required to achieve an appropriate sample size.
In additional cases of diseases, disorders, or conditions where there are no
therapeutic alternatives development, sponsors may choose to expedite the
development
of the candidate therapeutic intervention without making use of the above FDA
regulatory clinical development incentives. In these cases, the sponsor
proposes
expedited clinical development of a candidate therapeutic intervention due to
outstanding
positive or unequivocal preclinical safety and/or efficacy data.
As used herein, "supplemental applications" are those in which a candidate
therapeutic intervention is tested in a human clinical trial in order for the
product to have
an expanded label to include additional indications for therapeutic use. In
these cases, the
previous clinical studies of the therapeutic intervention, i.e. those
involving the
preclinical safety and Phase I human safety studies can be used to support the
testing of
the particular candidate therapeutic intervention in a patient population for
a different
disease, disorder, or condition than that previously approved in the US. In
these cases, a
limited Phase II study is performed in the proposed patient population. With
adequate
signs of efficacy, a Phase III study is designed. All other parameters of
clinical
development for this category of candidate therapeutic interventions proceeds
as
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
described above for interventions first tested in human candidates.
As used herein, "outcomes" or "therapeutic outcomes" are used to describe the
results and value of healthcare intervention. Outcomes can be mufti-
dimensional, e.g.,
including one or more of the following: improvement of symptoms; regression of
the
disease, disorder, or condition; economic outcomes of healthcare decisions.
As used herein, "pharmacoeconomics" is the analysis of a therapeutic
intervention in a population of patients diagnosed with a disease, disorder,
or condition
that includes at least one of the following studies: cost of illness study
(COn; cost
benefit analysis (CBA), cost minimization analysis (CMA), or cost utility
analysis
1o (CUA), or an analysis comparing the relative costs of a therapeutic
intervention with one
or a group of other therapeutic interventions. In each of these studies, the
cost of the
treatment of a disease, disorder, or condition is compared among treatment
groups. As
used herein, costs are those economic variables associated with a disease,
disorder, or
condition fall into two broad categories: direct and indirect. Direct costs
are associated
with the medical and non-medical resources used as therapeutic interventions,
including
medical, surgical, diagnostic, phannacologic, devices, rehabilitation, home
care, nursing
home care, institutional care, and prosthesis. Indirect costs are associated
with loss of
productivity due to the disease, disorder, or condition suffered by the
patient or relatives.
A third category, the tangible and intangible losses due to pain and suffering
of a patient
or relatives oRen is included in indirect cost studies.
As used herein, "health-related quality of life" is a measure of the impact of
the
disease, disorder, or condition on an individual's or group of patient's
activities of daily
living. Preferably, included in pharmacoeconomic studies is an analysis of the
health-
related quality of life. Standardized surveys or questionnaires for general
health-related
quality of life or disease, disorder, or condition specific determine the
impact the disease,
disorder, or condition has on an individuals day to day life activities or
specific activities
that are affected by a particular disease, disorder, or condition.
As used herein, the term "stratification" refers to the creation of a
distinction
between patients on the basis of a characteristic or characteristics of the
patient.
3o Generally, in the context of clinical trials, the distinction is used to
distinguish responses
or effects in different sets of patients distinguished according to the
stratification
parameters. For the present invention, stratification preferably includes
distinction of
patient groups based on the presence or absence of particular variance or
variances in one
or more genes. The stratification may be performed only in the course of
analysis or may
be used in creation of distinct groups or in other ways.
A human clinical trial can result in data to support the utility of a gene
variance or
variances for the selection of optimal therapy. Clinical studies require no
knowledge of
the biological function of the gene containing the variance of the variances
to be assessed,
CA 02335649 2001-O1-19
71
WO 00/04194 PCT/US99/16440
nor any knowledge of how the therapeutic invention to be assessed works at a
biochemical level.
There are several important preclinical data sets that pose criteria to
consider
when designing a clinical study to assess the utility of a variance in a gene
for selecting
optimal therapy for a disease, disorder, or condition. Preferably, the data
sets include one
or a combination of at least of the following:
Mechanism of action of the therapeutic intervention-
If the candidate therapy (e.g. drug) has established mechanism of action, the
target genes
can be appropriately identified. In vitro data supporting altered physiologic
activity of
the variant forms of the gene in the presence of the therapy, assists the
direction of the
fundamental hypotheses and identifying the objectives for a human clinical
trial.
Mechanism of metabolic transformation of the therapeutic intervention-
15 If in vitro or in vivo animal studies have demonstrated metabolic
biotransformation of the
therapeutic intervention, correlation of the effects of a variance or
variances on the
metabolic biotransformation of the therapeutic intervention can further assist
the direction
of the fundamental hypotheses and identification of the objectives of the
human clinical
study.
Effect of the variance or variances on therapeutic intervention-
The combined preclinical data sets should point to the premise of a controlled
clinical
trial of the the therapeutic intervention. The design of the trial will
preferably incorporate
the preclinical data sets to determine the primary and secondary endpoints.
Preferably,
these endpoints will include whether the therapeutic intervention is
efficacious,
efficacious with undesirable side effects, ineffective, ineffective with
undesirable side
effects, or ineffective with deleterious effects. Pharmacoeconomic analyses
may be
incorporated in order to support the efficacious intervention, efficacious
with undesirable
side effects cases, whereby the clinical outcome is positive, and economic
analyses are
required for the support of overall benefit to the patient and to society.
The strategies for designing a clinical trial to test the effect of a
genotypic
variance or variances on a physiological response to therapeutic intervention
for drugs
with known mechanism of action, mechanism of biotransformation, and/or known
physiologic response differentials correlated to genotvpic variance or
variances will be
modified based upon the data and information from the preclinical studies and
the patient
symptomatic parameters unique to the target indication. However, the strategy
(design)
and the implementation (conduct) of the clinical study preferably consist of
one or more
of the following strategies.
CA 02335649 2001-O1-19
72
WO 00/04194 PCT/US99/16440
A. Retrospective clinical trials.
In general the goal of retrospective clinical trials will be to test and
refine
hypotheses regarding genetic factors that are associated with drug responses.
The best
supported hypotheses can subsequently be tested in prospective clinical
trials, and data
from the prospective trials will likely comprise the main basis for an
application to
register the drug and predictive genetic test with the appropriate regulatory
body. In
some cases, however, it may become acceptable to use data from retrospective
trials to
support regulatory filings.
I. Clinical trials to study the effect of one gene locus on drug response
A. Stratify patients by genotype at one candidate variance in the candidate
gene locus.
1. Genetic stratification of patients can be accomplished in several
ways, including the following (where 'A' is the more frequent form of the
variance being assessed and 'a' is the less frequent form):
(a) AA vs. as
(b) AA vs. Aa vs. as
(c) AA vs. (Aa + aa)
(d) (AA + Aa) vs. aa.
2. The effect of genotype on drug response phenotype may be
affected by a variety of nongenetic factors. Therefore it may be beneficial to
measure the effect of genetic stratification in a subgroup of the overall
clinical trial population. Subgroups can be defined in a number of ways
including, for example, biological, clinical, pathological or environmental
criteria. For example, the predictive value of genetic stratification can be
assessed in a subgroup or subgroups defined by:
a. Biological criteria:
i. gender (males vs. females)
ii. age (for example above 60 years of age). Two, three or more
age groups may be useful for defining subgroups for the genetic analysis.
iii. hormonal status and reproductive history, including pre- vs.
post- menopausal status of women, or multiparous vs. nulliparous women
iv. ethnic, racial or geographic origin, or surrogate markers of
ethnic, racial or geographic origin. (For a description of genetic markers
that
serve as surrogates of racial/thnic origin see, for example: Rannala, B. and
J.L. Mountain, Detecting immigration by using multilocus genotypes. Proc
CA 02335649 2001-O1-19
73
WO 00/04194 PCT/US99/1b440
Natl Acad Sci U S A , 94 (17): 9197-9201, 1997. Other surrogate markers
could be used, including biochemical markers.)
b. Clinical criteria:
i. Disease status. There are clinical grading scales for many
diseases. For example, the status of Alzheimer's Disease patients is often
measured by cognitive assessment scales such as the mini-mental status
exam (MMSE) or the Alzheimer's Disease Assessment Scale (ADAS),
which includes a cognitive component (ADAS-COG). There are also clinical
assessment scales for many other diseases, including cancer.
ii. Disease manifestations (clinical presentation).
c. Pathological criteria:
i. Histopathologic features of disease tissue, or pathological
diagnosis. (For example there are many varieties of lung cancer: squamous
cell carcinoma, adenocarcinoma, small cell carcinoma, bronchoalveolar
t5 carcinoma, etc., each of which may - which, in combination with genetic
variation, may correlate with
ii. Pathological stage. A variety of diseases have pathological
staging schemes
iii. Loss of heterozygosity (LOH)
2o iv. Pathology studies such as measuring levels of a marker
protein
v. Laboratory studies such as hormone levels, protein levels,
small molecule levels
25 3. Measure frequency of responders in each genetic subgroup.
Subgroups may be defined in several ways.
i. more than two age groups
ii. age related status such as pre or post-menopausal
Stratify by hapiotype at one candidate locus where the haplotype is made up
3o of two variances, three variances or greater than three variances.
4. Statistical analysis of clinical trial data
There are a variety of statistical methods for measuring the difference
between two or more groups in a clinical trial. One skilled in the art will
recognize
35 that different methods are suited to different data sets. In general, there
is a family
of methods customarily used in clinical trials, and another family of methods
customarily used in genetic epidemiological studies. Methods from either
family
CA 02335649 2001-O1-19
74
WO 00/04194 PCT/US99/16440
may be suitable for performing statistical analysis of pharmacogenetic
clinical trial
data.
a Conventionals'linical Trial ~tatict»c
Conventional clinical trial statistics include hypothesis testing and
descriptive methods, as elaborated below. Guidance in the selection of
appropriate
statistical tests for a particular data set can be obtained from texts such
as:
Biostatistics~ A Foundation for An~l_vsis in the Heg] ~ciencPC, 7th edition
(Wiley
Series in Probability and Mathematical Statistics, Applied Probability and
statistics)
1o by Wayne W. Daniel, John Wiley & Sons, 1998; Bavesia_n Methods a_nd Ethics
in a
Clinical Trial Design (Wiley Series in Probability and Mathematical
Statistics.
Applied Probability Section) by J. B. Kadane (Editor), John Wiley & Sons,
1996;
b. Hv~~othesis testing statistical procedures
(1) One-sample procedures (binomial confidence interval, Wilcoxon
signed rank test, permutation test with general scores, generation of exact
permutational distributions)
(2) Two-sample procedures (t-test, Wilcoxon-Mann-Whitney test,
2o Normal score test, Median test, Van der Waerden test, Savage test, Logrank
test for
censored survival data, Wilcoxon-Gehan test for censored survival data,
Cochran-
Armitage trend test, permutation test with general scores, generation of exact
permutational distributions)
(3) R x C contingency tables (Fisher's exact test, Pearson's chi-squared
test, Likelihood ratio test, Kruskal-Wallis test, Jonckheere-Terpstra test,
Linear-by
linear association test, McNemar's test, marginal homogeneity test for matched
pairs)
3o (4) Stratified 2 x 2 contingency tables (test of homogeneity for odds
ratio, test of unity for the common odds ratio, confidence interval for the
common
odds ratio)
(5) Stratified 2 x C contingency tables (all two-sample procedures listed
above with stratification, confidence intervals for the odds ratios and trend,
generation of exact permutational distributions)
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/Ib440
(6) General linear models (simple regression, multiple regression,
analysis of variance -ANOVA-, analysis of covariance, response-surface models,
weighted regression, polynomial regression, partial correlation, multiple
analysis of
variance -MANOVA-, repeated measures analysis of variance).
5
(7) Analysis of variance and covariance with a nested (hierarchical)
structure.
(8)~ Designs and randomized plans for nested and crossed experiments
to (completely randomized design for two treatment, split-splot design,
hierarchical
design, incomplete block design, Latin square design)
(9) Nonlinear regression models
I5 (10) Logistic regression for unstratified or stratified data, for binary or
ordinal response data, using the Iogit link function, the normit function or
the
complementary log-log function.
( 11 ) Probit, logit, ordinal logistic and gompit regression models.
( 12) Fitting parametric models to failure time data that may be right-, left-
,
or interval-censored. Tested distributions can include extreme value, normal
and
logistic distributions, and, by using a log transformation, exponential,
Weibull,
lognormal, loglogistic and gamma distributions.
(13) Compute non-parametric estimates of survival distribution with right-
censored data and compute rank tests for association of the response variable
with
other variables.
3o c. Descriptive statistical methods
~ Factor analysis with rotations
~ Canonical correlation
~ Principal component analysis for quantitative variables.
~ Principal component analysis for qualitative data.
~ Hierarchical and dynamic clustering methods to create tree structure,
dendrogram or phenogram.
~ Simple and multiple correspondence analysis using a contingency table
as input or raw categorical data.
CA 02335649 2001-O1-19
76
WO 00/04194 PCT/US99/16440
Specific instructions and computer programs for performing the above
calculations can be obtained from companies such as: SAS/STAT Software, SAS
Institute Inc., Cary, NC, USA; BMDP Statistical Software, BMDP Statistical
Software Inc., Los Angeles, CA, USA; SYSTAT software, SPSS Inc., Chicago, IL,
USA; StatXact & LogXact, CYTEL Software Corporation, Cambridge, MA, USA.
d. Statistical Methods from Genetic Epidemioloev
Genetic epidemiological methods can also be useful in carrying out statistical
to tests for the present invention.
Guidance in the selection of appropriate genetic statistical tests for
analysis
of a particular data set can be obtained from texts such as: Fundamentals of
Genetic
Epidemioloev (Monographs in Epidemiology and Biostatistics, Vol 22) by M. J.
Khoury, B. H. Cohen & T. H. Beaty, Oxford Univ Press, 1993; Methods in Genetic
15 Enidemiolo~y by Newton E. Morton, S. Karger Publishing, 1983; Methods in
Observational ~pidemiolo-gv, 2nd edition (Monographs in Epidemiology and
Biostatistics, V. 26) by J. L. Kelsey (Editor), A. S. Whittemore & A. S.
Evans, 1996;
Clinical Trials ~ DesiQn Conduct and Analysis (Monographs in Epidemiology and
Biostatistics, Vol 8) by C. L. Meinert & S. Tonascia, 1986)
Strategy for the implementation of a clinical study in the case of a
therapeutic with known mechanism of action:
1. Identify genes that encode proteins that perform functions related to drug
absorption and/or, distribution, as well as genes related to the
pharmacological
action (pharmacodynamics) of the therapeutic intervention. Genes that encode
proteins homologous to the proteins believed to carry out the above functions
are
also worth evaluation as they may carry out similar functions. Together the
foregoing proteins constitute the candidate genes for affecting response of a
patient
to the therapeutic intervention.
2. Identify variances in the candidate genes. Initially, individual variances
(and
preferably their frequencies) will be identified by standard methods. Then,
for genes
with more than one variance, the commonly occurring patterns of variances
occurring on a single chromosome (i.e. the haplotypes) may also be established
using both computational and experimental approaches. For example, a
computational approach might include one of, but not limited to, the following
two
methods a) expectation maximization (E-M) algorithm (Excoffier and Slatkin,
Mol.
Biol. Evol. 1995) and, b) a combination of Parsimonious and E-M methods.
CA 02335649 2001-O1-19
77
WO 00/04194 PCT/US99/16440
If we have a large population, implementation of the E-M method will be
performed first.
A given phenotype or a sequence could come from several genotypes. This
is particularly true if the sequence is heterozygous at a number of nucleotide
positions. Therefore, it is not practical to just count the phenotypes and
make a
conclusion on the underlying genotype, because it may lead to ambiguities. To
avoid such ambiguities, an alternative iterative method called the EM
(expectation-
maximization) algorithm is used to derive the expected genotypes for a given
phenotype or a sequence. This method assumes that the population under
consideration is in Hardy-Weinberg equilibrium.
For example, consider the ABO locus in a population. Supposing , there are
Na people of type A, Nb people of type B, Nab people of type AB, and No people
of
type O. Assuming N = Na + Nb + Nab + No in the random sample of people N, we
cannot tell exactly how many of the Na people are homozygous for AlA and how
t 5 many are heterozygotes for AlO.
In order to avoid this dilemma, we first assume that the expected number of
genotypic frequencies in the population is in H-W equilibrium for any given
(all)
alleles) frequency. This is followed by setting the allele frequencies and
iteration n,
and testing for its stability in a series of iterations, up to m. When the
values of the
initial allele frequencies stabilize at the end of series of iterations up to
m, the
resulting expected number of genotypes are assigned to phenotypes; for
example,
sequences or individuals.
The following steps are involved in the E-M algorithm:
Chose an allele or a haplotype in an expected class that occurs at the highest
frequency
2. Use it as a base for the observed values and estimate the unobserved or the
expected value
3. Use the second value as the true value and estimate the unobserved value
from
the second value
304. Continue this process (up to m) till you find values that do not change
from one
iteration to the next.
5. The final value is the maximum likelihood (highly likely) estimate of that
allele
or the haplotype
As indicated above, also among the number of methods which are used for
the purpose of classifying DNA sequences, haplotypes or phenotypic characters
are
the parsimony methods. Parsimony principle maintains that the best explanation
for
CA 02335649 2001-O1-19
WO 00/04194 PCT/LJS99/16440
the observed differences among sequences, phenotypes (individuals, species)
etc., is
provided by the smallest number of evolutionary changes. Alternatively,
simpler
hypotheses are preferable to explain a set of data or patterns, than more
complicated
ones, and that ad hoc hypotheses should be avoided whenever possible
(Molecular
Systematics, Hillis et al., 1996). These methods for inferring relationship
among
sequences operate by minimizing the number of evolutionary steps or mutations
(changes from one sequence/character) required to explain a given set of data.
For example, supposing we want to obtain relationships among a set of
sequences and construct a structure (tree/topology), we first count the
minimum
to number of mutations that are required for explaining the observed
evolutionary
changes among a set of sequences. A structure (topology) is constructed based
on
this number. When once this number is obtained, another structure is tried.
This
process is continued for all reasonable number of structures. Finally, the
structure
that required the smallest number of mutational steps is chosen as the likely
structure/evolutionary tree for the sequences studied.
If the computed frequency of the haplotypes are equal to the number of
individuals in the population, then there will be a consideration of utilizing
additional methods. For these cases and if there is a small population, then
the
number of haplotypes will be considered relative to the number of entrants. In
a
method that is a modification of previously published work (Clark, Mol Biol
and
Evol. 1990) homozygotes will be assigned one unambiguous haplotype. If there
is a
single site variance (mutation) at one of the chromosomes then it will have
two
haplotypes. As the number of variances (mutations) increase in the diploid
chromosomes, each of these variances will be compared with the haplotypes of
the
original population. Then a frequency will be assigned to the new variance
based
upon the Hardy-Weinberg expected frequencies. (See text below for why
haplotypes
are useful and how to determine them experimentally, if necessary.)
3. Retrospectively reanalyze data from already completed clinical trials.
Since
3o the questions are new, the data can be treated as if it were a prospective
trial, with
identified variances or haplotypes as stratification criteria and
biological/clinical
endpoints. Care should be taken to avoid studying a population in which there
may
be a link between drug-related genes and disease-related genes.
4. Select group of variances or haplotypes to differentiate: one control group
including groups of variances with normal biological response one or a few
case
groups including groups of variances with significant biological impact
5. Establish phase III trials with selected variances as inclusion criteria
and
clinical/pharmacoeconomic endpoints. The number of patients required for
adequate
CA 02335649 2001-O1-19
79
WO 00/04194 PCT/US99/16440
statistical power (approximately the same as in a usual phase III trial) will
be
determined from the phase II results and allele frequencies.
Strategy for the implementation of a clinical study in the case of a
therapeutic intervention with known mechanism of biotransformation:
I . Identify genes that encode proteins that perform functions related to drug
biotransformation or excretion, as well as genes related to the
pharmacological
action (pharmacodynamics) of the metabolized or biotransformed therapeutic
intervention. Genes that encode proteins homologous to the proteins believed
to
carry out the above functions are also worth evaluation as they may carry out
similar
functions. Together the foregoing proteins constitute candidate genes for
affecting
response of a patient to the therapeutic intervention.
2. Identify variances in the candidate genes. Initially, individual variances
will
be identified by standard methods. Then, for genes with more than one
variance, the
t5 commonly occurring patterns of variances occurring on a single chromosome
(i.e.
the haplotypes) may also be established. (See text below for why haplotypes
are
useful and how to determine them experimentally, if necessary.)
3. Retrospectively reanalyze data from already completed clinical trials.
Since
the questions are new, the data can be treated as if it were a prospective
trial, with
2o identified variances or haplotypes as stratification criteria and
biologicaUclinical
endpoints. Care should be taken to avoid studying a population in which there
may
be a link between drug-related genes and disease-related genes.
4. Select group of variances or haplotypes to differentiate: one control group
including groups of variances with normal biological response one or a few
case
25 groups including groups of variances with significant biological impact.
5. Establish phase III trials with selected variances as inclusion criteria
and
clinicaUpharmacoeconomic endpoints. The number of patients required for
adequate
statistical power (approximately the same as in a usual phase III trial) will
be
determined from the phase II results and allele frequencies.
Strategy for the implementation of a clinical study in the case of a
therapeutic intervention where by the effect of the gene variance or variances
on
therapeutic intervention is known:
1. Retrospectively reanalyze data from already completed clinical trials. In
this
case, since the questions are new, the data can be treated as if it were a
prospective
trial, with identified variances or haplotypes as stratification criteria and
biological/clinical endpoints. Care should be taken to avoid studying a
population in
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
which there may be a link between drug-related genes and disease-related
genes.
2. Select group of variances or haplotypes to differentiate: one control group
including groups of variances with normal biological response and one or a few
case
groups including groups of variances with significant biological impact.
3. Establish phase III or phase IV {post marketing) trials with selected
variances
as inclusion criteria and clinicaUpharmacoeconomic endpoints. The number of
patients required for adequate statistical power (approximately the same as in
a usual
phase III trial) will be determined from the phase II results and allele
frequencies.
1o A clinical trial in which phatmacogenetic related efficacy or toxicity
endpoints are included in the primary or secondary endpoints will be part of a
retrospective or prospective clinical trial. In the design of these trials,
the allelic
differences will be identified and stratification based upon these genotypic
differences among patient or subject groups will be used to ascertain the
significance
15 of the impact a genotype has on the candidate therapeutic intervention.
Retrospective pharmacogenetic trials can be conducted at each of the phases of
clinical development, with the assumption that sufficient data is available
for the
correlation of the physiologic effect of the candidate therapeutic
intervention and the
allelic variance or variances within the treatment population. In the case of
a
2o retrospective trial, the data collected from the trial can be re-analyzed
by imposing
the additional stratification on groups of patients by specific allelic
variances that
may exist in the treatment groups. Retrospective trials can be useful to
ascertain
whether a hypothesis that a specific variance has a significant effect on the
efficacy
or toxicity profile for a candidate therapeutic intervention.
25 A prospective clinical trial has the advantage that the trial can be
designed to
ensure the trial objectives can be met with statistical certainty. In these
cases, power
analysis, which includes the parameters of allelic variance frequency, number
of
treatment groups, and ability to detect positive outcomes can ensure that the
trial
objectives are met.
30 In designing a pharmacogenetic trial, retrospective analysis of Phase II or
Phase III clinical data can indicate trial variables for which further
analysis is
required. For example, surrogate endpoints, pharmacokinetic parameters,
dosage,
efficacy endpoints, ethnic and gender differences, and toxicological
parameters may
result in data that would require further analysis and re-examination through
the
35 design of an additional trial. In these cases, analysis involving
statistics" genetics,
clinical outcomes, and economic parameters may be considered prior to
proceeding
to the stage of designing any additional trials. Factors involved in the
consideration
of statistical significance may include Bonferroni analysis, permutation
testing, with
CA 02335649 2001-O1-19
81
WO 00/04194 PCT/US99/1~6440
multiple testing correction resulting in a difference among the treatment
groups that
has occurred as a result of a chance of no greater than 20%, i.e. p< 0.20.
Factors
included in determining clinical outcomes to be relevant for additional
testing may
include, for example, consideration of the target indication, the trial
endpoints,
progression of the disease, disorder, or condition during the trial study
period,
biochemical or pathophysiologic relevance of the candidate therapeutic
intervention,
and other variables that were not included or anticipated in the initial study
design or
clinical protocol. Factors to be included in the economic significance in
determining
additional testing parameters include sample size, accrual rate, number of
clinical
to sites or institutions required, additional or other available medical or
therapeutic
interventions approved for human use, and additional or other available
medical or
therapeutic interventions concurrently or anticipated to enter human clinical
testing.
Further, there may be patients within the treatment categories that present
data that
fall outside of the average or mean values, or there may be an indication of
multiple
allelic loci that are involved in the responses to the candidate therapeutic
intervention. In these cases, one could propose a prospective clinical trial
having an
objective to determine the significance of the variable or parameter and its
effect on
the outcome of the parent Phase II trial. In the case of a pharmacogenetic
difference,
i.e. a single or multiple allelic difference, a population could be selected
based upon
2o the distribution of genotypes. The candidate therapeutic intervention could
then be
tested in this group of volunteers to test for efficacy or toxicity. The
repeat
prospective study could be a Phase I limited study in which the subjects would
be
healthy human volunteers, or a Phase II limited efficacy study in which
patients
which satisfy the inclusion criteria could be enrolled. In either case, the
second,
confirmatory trial could then be used to systematically ensure an adequate
number of
patients with appropriate phenotype is enrolled in a Phase III trial.
A placebo controlled pharmacogenetics clinical trial design will be one in
which target allelic variance or variances will be identified and a diagnostic
test will
be performed to stratify the patients based upon presence, absence, or
combination
3o thereof of these variances. In the Phase II or Phase III stage of clinical
development,
determination of a specific sample size of a prospective trial will be
described to
include factors such as expected differences between a placebo and treatment
on the
primary or secondary endpoints and a consideration of the allelic frequencies.
The design of a pharmacogenetics clinical trial will include a description of
the allelic variance impact on the observed efficacy between the treatment
groups.
Using this type of design, the type of genetic and phenotypic relationship
display of
the efficacy response to a candidate therapeutic intervention will be
analyzed. For
example, a genotypically dominant allelic variance or variances will be those
in
CA 02335649 2001-O1-19
82
WO 00/04194 PCT/US99/16440
which both heterozygotes and homozygotes will demonstrate a specific
phenotypic
efficacy response different from the homozygous recessive genotypic group. A
phannacogenetic approach is useful for clinicians and public health
professionals to
include or eliminate small groups of responders or non-responders from
treatment in
order to avoid unjustified side-effects. Further, adjustment of dosages when
clear
clinical difference between heterozygous and homozygous individuals may be
beneficial for therapy with the candidate therapeutic intervention
In another example, a reccesive allelic variance or variances will be those in
which only the homozygote recessive for that or those variances will
demonstrate a
1o specific phenotypic efficacy response different from the heterozygotes or
homozygous dominants. An extension of these examples may include allelic
variance or variances organized by haplotypes from additional gene or genes
providing an explanation of clinical phenotypic outcome differences among the
treatment groups. These types of clinical studies will point and address
allelic
variance and its role in the efficacy or toxicology pattern within the
treatment
population.
IV. Variance Identification and Use
A. Initial Identification of variances in genes
Selection of population size and composition
Prior to testing to identify the presence of sequence variances in a
particular gene or genes, it is useful to understand how many individuals
should be screened to provide confidence that most or nearly all
pharmacogenetically relevant variances will be found. The answer depends
on the frequencies of the phenotypes of interest and what assumptions we
make about heterogeneity and magnitude of genetic effects. At the
beginning we only know phenotype frequencies (e.g. responders vs.
nonresponders, frequency of various side effects, etc.). As an example, the
occurrence of serious 5-FU/FA toxicity - e.g. toxicity requiring
3o hospitalization is often >10%. The occurrence of life threatening toxicity
is
in the 1-3% range (Buroker et al. 1994). The occurrence of complete
remissions is on the order of 2-8%, The lowest frequency phenotypes are
thus on the order of ~2%. If we assume that (i) homogeneous genetic effects
are responsible for half the phenotypes of interest and (ii) for the most part
the extreme phenotypes represent recessive genotypes, then we need to detect
alleles that will be present at --10% frequency (.l x .1 = .01, or 1%
frequency
of homozygotes) if the population is at Hardy-Weinberg equilibrium. To
have a ~99% chance of identifying such alleles would require searching a
CA 02335649 2001-O1-19
83
WO 00/04194 PCT/US99/16440
population of 22 individuals (see Table 1 below). If the major phenotypes
are associated with heterozygous genotypes then we need to detect alleles
present at ~.5% frequency (2 x .005 x .995 = .00995, or ~l% frequency of
heterozygotes). A 99% chance of detecting such alleles would require ~40
individuals (Table below). Given the heterogeneity of the North American
population we cannot assume that all genotypes are present in Hardy-
Weinberg proportions, therefore a substantial oversampling is done to
increase the chances of detecting relevant variances: For our initial
screening,
usually, 62 individuals of known race/ethnicity are screened for variance.
to Variance detection studies can be extended to outliers for the phenotypes
of
interest to cover the possibility that important variances were missed in the
normal population screening.
Table 1
Number otyped
of
subjects
gen
Allele n=5 n= n= n=20 n=25 n=30 n=35 n=50
fre uencies 10 15
p=.99, 9.56 18.21 26.0333.10 39.50 45.28 50.52 63.40
%
p=.97, 26.26 45.62 59.9070.43 78.19 83.92 88.14 95.24
p=.95, 40.13 64.15 78.5387.15 92.30 95.39 97.24 99.65
p=.93, 51.60 76.58 88.6694.51 97.34 98.71 99.38 99.93
p =.9, 65.13 87.84 95.7698.52 99.48 99.82 99.94 >99.9
q =
p =.8, 89.26 98.84 99.8899.99 >99.9 >99.99>99.9 >99.9
q =
p =.7, 97.17 99.92 99.99>99.99>99.9 >99.99>99.9 >99.9
q =
is
Likelihood of Detecting Polymorphism in a Population as a Function
of Allele Frequency do Number of Individuals Genotyped
2o The table above shows the probability (expressed as percent) of
detecting both alleles (i.e. detecting heterozygotes) at a biallelic locus as
a
function of (i) the allele frequencies and (ii) the number of individuals
genotyped. The chances of detecting heterozygotes increases as the
frequencies of the two alleles approach 0.5 (down a column), and as the
25 number of individuals genotyped increases (to the right along a row). The
numbers in the table are given by the formula: 1 - (p)2n - (q)2n. Allele
frequencies are designated p and q and the number of individuals tested is
designated n. (Since humans are diploid, the number of alleles tested is
twice the number of individuals, or 2n.)
CA 02335649 2001-O1-19
WO 00/04194 ~ PCT/1JS99/16A40
While it is preferable that numbers of individuals, or independent
sequence samples, are screened to identify variances in a gene, it is also
very
beneficial to identify variances using smaller numbers of individuals or
sequence samples. For example, even a comparison between the sequences
of two samples or individuals can reveal sequence variances between them.
Preferably, 5, 10, or more samples or individuals are screened.
Source of nucleic acid samples
Nucleic acid samples, for example for use in variance identification,
1o can be obtained from a variety of sources as known to those skilled in the
art,
or can be obtained from genomic or cDNA sources by known methods. For
example, the Coriell Cell Repository (Camden, N.J.) maintains over 6,000
human cell cultures, mostly fibroblast and lymphoblast cell lines comprising
the NIGMS Human Genetic Mutant Cell Repository. A catalog
15 (http://locus.umdnj.edulnigms) provides racial or ethnic identifiers for
many
of the cell lines. 55 of the 62 cell lines to be genotyped (as indicated
above)
are drawn from this collection; the remainder were obtained from the Beijing
Cancer Institute. The cell lines are derived from 21 Caucasians (of Northern,
Central and Southern European origin), 8 Afro-Americans, 9 Hispanics or
20 Mexicans, 8 Chinese, 12 Japanese, 1 American Indian, 1 East Indian, 1
Iranian, and 1 Korean. These cell lines (plus ~75 other lymphoblastoid lines)
are currently in use by the inventors for variance detection studies.
Source of human DNA, RNA and cDNA samples
z5 PCR based screening for DNA polymorphism can be carried out
using either genomic DNA or cDNA produced from mRNA. For many
genes, only cDNA sequences have been published, therefore the analysis of
those genes is, at least initially, at the cDNA level since the detenmination
of
intron-exon boundaries and the isolation of flanking sequences is a laborious
3o process. However, screening genomic DNA has the advantage that variances
can be identified in promoter, intron and flanking regions. Such variances
may be biologically relevant. Therefore preferably, when variance analysis
of patients with outlier responses is performed, analysis of selected loci at
the
genomic level is also performed. Such analysis would be contingent on the
35 availability of a genomic sequence or intron-exon boundary sequences, and
would also depend on the anticipated biological importance of the gene in
connection with the particular response.
When cDNA is to be analyzed it is very beneficial to establish a
CA 02335649 2001-O1-19
WO 00/04194
PCT/US99/16d40
tissue source in which the genes of interest are expressed at sufficient
levels
that cDNA can be readily produced by RT-PCR. Preliminary PCR
optimization efforts for 19 of the 29 genes in Table 2 reveal that all 19 can
be
amplified from lymphoblastoid cell mRNA. The 7 untested genes belong on
s the same pathways and are expected to also be >rCR amplifable.
PCR Optimization
Primers for amplifying a particular sequence can be designed by
methods known to those skilled in the art, including by the use of computer
to programs such as the PRIMER software available from Whitehead
Institute/MIT Genome Center. In some cases it is preferable to optimize the
amplification process according to parameters and methods known to chose
skilled in the .art; optimization of PCR reactions based on a limited array of
temperature, buffer and primer concentration conditions is utilized. New
is primers are obtained if optimization fails with a particular primer set.
Variance detection using T4 endonuclease VII mismatch cleavage
method
Any of a variety of different methods for detecting variances in a
2o particular gene can be utilized, such as those described in the patents and
applications cited in section A above. An exemplary method is a T4
EndoVII method. The enzyme T4 endonuclease VII (T4E7) is derived from
the bacteriophage T4. T4E7 specifically cleaves heteroduplex DNA
containing single base mismatches, deletions or insertions. The site of
25 cleavage is 1 to 6 nucleotides 3' of the mismatch. This activity has been
exploited to develop a general method for detecting DNA sequence variances
(Youil et al. 1995; Mashal and Sklar, 1995). A quality controlled T4E7
variance detection procedure based on the T4E7 patent of R.G.H. Cotton and
co-workers. (Del Tito et al., in press) is preferably utilized. T4E7 has the
30 advantages of being rapid, inexpensive, sensitive and selective. Further,
since the enzyme pinpoints the site of sequence variation, sequencing effort
can be confined to a 25 -30 nucleotide segment.
The major steps in identifying sequence variations in candidate genes
using T4E7 are: ( 1 ) PCR amplify 400-600 by segments from a panel of DNA
35 samples; (2) mix a fluorescently-labeled probe DNA with the sample DNA;
(3) heat and cool the samples to allow the formation of heteroduplexes; (4)
add T4E7 enzyme to the samples and incubate for 30 minutes at 3'7oC,
during which cleavage occurs at sequence variance mismatches; (5) run the
CA 02335649 2001-O1-19
86
WO 00/04194 PCT/US99/1G.440
samples on an ABI 377 sequencing apparatus to identify cleavage bands,
which indicate the presence and location of variances in the sequence; (6) a
subset of PCR fragments showing cleavage are sequenced to identify the
exact location and identity of each variance.
The T4E7 Variance Imaging procedure has been used to screen
particular genes. The efficiency of the T4E7 enzyme to recognize and cleave
at all mismatches has been tested and reported in the literature. One group
reported detection of 81 of 81 known mutations (Youil et al. 1995) while
another group reported detection of 16 of 17 known mutations (Mashal and
to Sklar, 1995). Thus, the T4E7 method provides highly efficient variance
detection.
DNA sequencing
A subset of the samples containing each unique T4E7 cleavage site is
t5 selected for sequencing. DNA sequencing can , for example, be performed
on ABI 377 automated DNA sequencers using BigDye chemistry and cycle
sequencing. Analysis of the sequencing runs will be limited to the 30-40
bases pinpointed by the T4E7 procedure as containing the variance. This
provides the rapid identification of the altered base or bases.
2o In some cases, the presence of variances can be inferred from
published articles which describe Restriction Fragment Length
Polymorphisms (RFLP). The sequence variances or polymorphisms creating
those RFLPs can be readily determined using convention techniques, for
example in the following manner. If the RFLP was initially discovered by
25 the hybridization of a cDNA, then the molecular sequence of the RFLP can
be determined by restricting the cDNA probe into fragments and separately
hybridizing to a Southern blot consisting of the restriction digestion with
the
enzyme which reveals the polymorphic site, identifying the sub-fragment
which hybridizes to the polymorphic restriction fragment, obtaining a
3o genomic clone of the gene (e.g., from commercial services such as Genome
Systems (Saint Louis, Missouri) or Research Genetics (Alabama) which will
provide appropriate genomic clones on receipt of appropriate primer pairs).
Using the genomic clone, restrict the genomic clone with the restriction
enzyme which revealed the polymorphism and isolate the fragment which
35 contains the polymorphism, e.g., identifying by hybridization to the cDNA
which detected the polymorphism. The fragment is then sequenced across
the polymorphic site. A copy of the other allele can be obtained by PCT
from addition samples.
CA 02335649 2001-O1-19
87
WO 00/04194 PCT/US99/16440
Variance detection using sequence scanning
In addition to the physical methods, e.g., those described above and
others known to those skilled in the art (see, e.g., Housman, U.S. Patent
5,702,890; Housman et al., U.S. Patent Application 09/045,053), variances
s can be detected using computational methods, involving computer
comparison of sequences from two or more different biological sources,
which can be obtained in various ways, for example from public sequence
databases. The term "variance scanning" refers to a process of identifying
sequence variances using computer-based comparison and analysis of
multiple representations of at least a portion of one or more genes.
Computational variance detection involves a process to distinguish true
variances from sequencing errors or other artifacts, and thus does not require
perfectly accurate sequences. Such scanning can be performed in a variety of
ways as known to those skilled in the art, preferably, for example, as
described in Stanton and Adams, U.S. Patent Application filed April 26,
1999, serial number not yet assigned, attorney docket 241/034.
While the utilization of complete cDNA sequences is highly preferred, it is
also possible to utilize genomic sequences. Such analysis may be desired where
the
detection of variances in or near splice sites is sought. Such sequences may
2o represent full or partial genomic DNA sequences for a gene or genes. Also,
as
previously indicated, partial cDNA sequences can also be utilized although
this is
less preferred. As described below, the variance scanning analysis can simply
utilize
sequence overlap regions, even from partial sequences. Also, while the present
description is provided by reference to DNA, e.g., cDNA, some sequences may be
2s provided as RNA sequences, e.g., mRNA sequences. Such RNA sequences may be
converted to the corresponding DNA sequences, or the analysis may use the RNA
sequences directly.
B. Determination of Presence or Absence of Known Variances
30 The identification of the presence of previously identified variances in
cells
of an individual, usually a particular patient, can be performed by a number
of
different techniques as indicated in the Summary above. Such methods include
methods utilizing a probe which specifically recognizes the presence of a
particular
nucleic acid or amino acid sequence in a sample. Common types of probes
include
3s nucleic acid hybridization probes and antibodies, for example, monoclonal
antibodies, which can differentially bind to nucleic acid sequences differing
in one
or more variance sites or to polypeptides which differ in one or more amino
acid
residues as a result of the nucleic acid sequence variance or variances.
Generation
CA 02335649 2001-O1-19
88
WO 00/04194 PCT/US99/16440
and use of such probes is well-known in the art and so is not described in
detail
herein.
Preferably, however, the presence or absence of a variance is determined
using nucleotide sequencing of a short sequence spanning a previously
identified
variance site. This will utilize validated genotyping assays for the
polymorphisms
previously identified. Since both normal and tumor cell genotypes can be
measured,
and since tumor material will frequently only be available as paraffin
embedded
sections (from which RNA cannot be isolated), it will be necessary to utilize
genotyping assays that will work on genomic DNA. Thus PCR reactions will be
to designed, optimized, and validated to accommodate the intron exon structure
of each
of the genes. If the gene structure has been published (as it has for some of
the listed
genes), PCR primers can be designed directly. However, if the gene structure
is
unknown, the PCR primers may need to be moved around in order to both span the
variance and avoid exon-intron boundaries. In some cases one-sided PCR methods
such as bubble PCR (Ausubel et al. 1997) may be useful to obtain flanking
intronic
DNA for sequence analysis.
Using such amplification procedures, the standard method used to genotype
normal and tumor tissues will be DNA sequencing. PCR fragments encompassing
the variances will be cycle sequenced on ABI 377 automated sequencers using
Big
2o Dye chemistry
C. Correlation of the Presence or Absence of Specific Variances with
Differential Treatment Response
Prior to establishment of a diagnostic test for use in the selection of a
treatment method or elimination of a treatment method, the presence or absence
of
one or more specific variances in a gene or in multiple genes is correlated
with a
differential treatment response. (As discussed above, usually the existence of
a
variable response and the correlation of such a response to a particular gene
is
performed first.) Such a differential response can be determined using
prospective
3o andlor retrospective data. Thus, in some cases, published reports will
indicate that
the course of treatment will vary depending on the presence or absence of
particular
variances. That information can be utilized to create a diagnostic test and/or
incorporated in a treatment method as an efficacy or safety determination
step.
Usually, however, the effect of one or more variances is separately
determined. The determination can be performed by analyzing the presence or
absence of particular variances in patients who have previously been treated
with a
particular treatment method, and correlating the variance presence or absence
with
the observed course, outcome, and/or development of adverse events in those
CA 02335649 2001-O1-19
89
WO 00/04194 PCT/US99/16440
patients. This approach is useful in cases where both the observation of
treatment
effects was clearly recorded and cell samples are available or can be
obtained.
Alternatively, the analysis can be performed prospectively, where the presence
or
absence of the variance or variances in an individual is determined and the
course,
outcome, and/or development of adverse events in those patients is
subsequently or
concurrently observed and then correlated with the variance determination.
Analysis of Haplotypes Increases Power of Genetic Analysis
Usually, variation in activity due to a single gene or a single genetic
variance
to in a single gene is not sufficient to account for observed variation in
patient response
to a treatment, e.g., a drug, there are often other factors that account for
some of the
variation in patient response. This is to be expected as drug response
phenotypes
usually vary continuously, and such (quantitative) traits are typically
influenced by a
number of genes (Falconer and Mackay, 1997). Although it is impossible to
t5 determine a priori the number of genes influencing a quantitative trait,
often only a
few loci have large effects, where a large effect is 5-20% of total variation
in the
phenotype (Mackay, 1995).
Having identified genetic variation in enzymes that may affect action of a
2o specific drug, it is useful to efficiently address its relation to
phenotypic variation.
The sequential testing for correlation between phenotypes of interest and
single
nucleotide polymorphisms may be adequate to detect associations if there are
major
effects associated with single nucleotide changes; certainly it is useful to
this type of
analysis. However there is no way to know in advance whether there are major
25 phenotypic effects associated with single nucleotide changes and, even if
thcre are,
there is no way to be sure that the salient variance has been identified by
screening
cDNAs. A more powerful way to address the question of genotype-phenotype
correlation is to assort genotypes into haplotypes. (A haplotype is the cis
arrangement of polymorphic nucleotides on a particular chromosome.) Haplotype
30 analysis has several advantages compared to the serial analysis of
individual
polymorphisms at a locus with multiple polymorphic sites.
(1 ) Of all the possible haplotypes at a locus (2" haplotypes are
theoretically possible at a locus with n binary polymorphic sites) only a
small
35 fraction will generally occur at a significant frequency in human
populations. Thus,
association studies of haplotypes and phenotypes will involve testing fewer
hypotheses. As a result there is a smaller probability of Type I errors, that
is, false
inferences that a particular variant is associated with a given phenotype.
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
(2) The biological effect of each variance at a locus may be different both
in magnitude and direction. For example, a polymorphism in the 5' UTR may
affect
translational efficiency, a coding sequence polymorphism may affect protein
5 activity, a polymorphism in the 3' LJTR may affect mRNA folding and half
life, and
so on. Further, there may be interactions between variances: two neighboring
poiymorphic amino acids in the same domain - say cys/arg at residue 29 and
met/val
at residue 166 - may, when combined in one sequence, for example, 29cys-
166va1,
have a deleterious effect, whereas 29cys-166met, 29arg-166met and 29arg-166va1
10 proteins may be nearly equal in activity. Haplotype analysis is the best
method for
assessing the interaction of variances at a locus.
(3) Templeton and colleagues have developed powerful methods for
assorting haplotypes and analyzing haplotype/phenotype associations (Templeton
et
t 5 al., 1987). Alleles which share common ancestry are arranged into a tree
structure
(cladogram) according to their time of origin in a population. Hapiotypes that
are
evolutionarily ancient will be at the center of the branching structure and
new ones
(reflecting recent mutations) will be represented at the periphery, with the
links
representing intermediate steps in evolution. The cladogram defines which
2o haplotype-phenotype association tests should be performed to most
efficiently
exploit the available degrees of freedom, focusing attention on those
comparisons
most likely to define functionally different haplotypes (Haviland et al.,
1995). This
type of analysis has been used to define interactions between heart disease
and the
apolipoprotein gene cluster {Haviland et al 1995) and Alzheimer's Disease and
the
25 Apo-E locus (Templeton 1995) among other studies, using populations as
small as
50 to 100 individuals.
Methods for determining haplotypes
The goal of haplotyping will be to identify the common haplotypes at
30 selected loci that have multiple sites of variance. Haplotypes will usually
be
determined at the cDNA level. Two general approaches to identification of
haplotyes will be employed. First, haplotypes will be inferred from the
pattern of
allele segregation in families collected by the Centre d'Etude Polymorphisme
Humaine. Cell lines from these families are available from the Coriell
Repository.
35 Cell lines for all members of families 884, 102, 104 and 1331 are currently
utilized.
Cell lines from six additional families will also be used to increase the
likelihood of
detecting common haplotypes. This approach will be useful for cataloging
common
haplotypes and for validating methods on samples with known haplotypes.
Second,
CA 02335649 2001-O1-19
9i
WO 00/04194 PCT/US99/16440
haplotypes will be determined directly from cDNA using the T4E7 procedure.
T4E7
cleaves mismatched heteroduplex DNA at the site of the mismatch. If a
heteroduplex contains only one mismatch, cleavage will result in the
generation of
two fragments. However, if a single heteroduplex (allele) contains two
mismatches,
cleavage will occur at two different sites resulting in the generation of
three
fragments. The appearance of a fragment whose size corresponds to the distance
between the two cleavage sites is diagnostic of the two mismatches being
present on
the same strand (allele). Thus, T4E7 can be used to determine haplotypes in
diploid
cells. -
1o An alternative method, allele specific PCR, may be used for haplotyping.
The utility of allele specific PCR for haplotyping has already been
established
(Michalatos-Beloin et al., 1996; Chang et al. 1997). Opposing PCR primers are
designed to coyer two sites of variance (either adjacent sites or sites
spanning one or
more internal variances). Two versions of each primer are synthesized,
identical to
15 each other except for the 3' terminal nucleotide. The 3' terminal
nucleotide is
designed so that it will hybridize to one but not the other variant base. PCR
amplification is then attempted with all four possible primer combinations in
separate wells. Because Taq polymerase is very inefficient at extending 3'
mismatches, the only samples which will be amplified will be the ones in which
the
20 two primers are perfectly matched for sequences on the same strand
(allele). The
presence or absence of PCR product allows haplotyping of diploid cell lines.
At
most two of four possible reactions should yield products. This procedure has
been
successfully applied, for example, to haplotype the DPD amino acid
polymorphisms.
25 D. Selection of Treatment Method Using Variance Information
1. General
Once the presence or absence of a variance or variances in a gene or genes is
shown to correlate with the efficacy or safety of a treatment method, that
information can be used to select an appropriate treatment method for a
particular
3o patient. In the case of a treatment which is more likely to be effective
when
administered to a patient who has at least one copy of a gene with a
particular
variance or variances (in some cases the correlation with effective treatment
is for
patients who are homozygous for variance or set of variances in a gene) than
in
patients with a different variance or set of variances, a method of treatment
is
35 selected (and/or a method of administration) which correlates positively
with the
particular variance presence or absence which provides the indication of
effectiveness. As indicated in the Summary, such selection can involve a
variety of
different choices, and the correlation can involve a variety of different
types of
CA 02335649 2001-O1-19
92
WO 00/04194 PCT/US99/16440
treatments, or choices of methods of treatment. In some cases, the selection
may
include choices between treatments or methods of administration where more
than
one method is likely to be effective, or where there is a range of expected
effectiveness or different expected levels of contra-indication or deleterious
effects.
In such cases the selection is preferably performed to select a treatment
which will
be as effective or more effective than other methods, while having a
comparatively
low level of deleterious effects. Similarly, where the selection is between
method
with differing levels of deleterious effects, preferably a method is selected
which has
low such effects but which is expected to be effective in the patient.
Alternatively, in cases where the presence or absence of the particular
variance or variances is indicative that a treatment or method of
administration is
more likely to be ineffective or contra-indicated in a patient with that
variance or
variances, then such treatment or method of administration is generally
eliminated
for use in that patient.
is
2. Diagnostic Methods
Once a correlation between the presence and absence of at least one variance
in a gene or genes and an indication of the effectiveness of a treatment, the
determination of the presence or absence of that at least one variance
provides
2o diagnostic methods, which can be used as indicated in the Summary above to
select
methods of treatment, methods of administration of a treatment, methods of
selecting a patient or patients for a treatment. and others aspects in which
the
determination of the presence or absence of those variances provides useful
information for selecting or designing or preparing methods or materials for
medical
25 use in the aspects of this invention. As previously stated, such variance
determination or diagnostic methods can be performed in various ways as
understood by those skilled in the art.
In certain variance determination methods, it is necessary or advantageous to
amplify one or more nucleotide sequences in one or more of the genes
identified
30 herein. Such amplification can be performed by conventional methods, e.g.,
using
polyrnerase chain reaction (PCR) amplification. Such amplification methods are
well-known to those skilled in the art and will not be specifically described
herein.
For most applications relevant to the present invention, a sequence to be
amplif ed
includes at least one variance site, which is preferably a site or sites which
provide
35 variance information indicative of the effectiveness of a method of
treatment or
method of administration of a treatment, or effectiveness of a second method
of
treatment which reduces a deleterious effect of a first treatment method, or
which
enhances the effectiveness of a first method of treatmcnt. Thus, for PCR, such
CA 02335649 2001-O1-19
93
WO 00/04194 PCT/US99/16440
amplification generally utilizes primer oligonucleotides which bind to or
extent
through at least one such variance site under amplification conditions.
For convenient use of the amplified sequence, e.g., for sequencing, it is
beneficial that the amplified sequence be of limited length, but still long
enough to
allow convenient and specific amplification. Thus, preferably the amplified
sequence has a length as described in the Summary.
Also, in certain variance determination, it is useful to sequence one or more
portions of a gene or genes, in particular, portions of the genes identified
in this
disclosure. As understood by persons familiar with nucleic acid sequencing. In
to particular, sequencing can utilize dye termination methods and mass
spectrometric
methods. The sequencing generally involves a nucleic acid sequence which
includes
a variance site as indicated above in connection with amplification. Such
sequencing can directly provide detenmination of the presence or absence of a
particular variance or set of variances, e.g., a haplotype, by inspection of
the
sequence (visually or by computer). Such sequencing is generally conducted on
PCR amplified sequences in order to provide sufficient signal for practical or
reliable sequence determination.
Likewise, in certain variance determinations, it is useful to utilize a probe
or
probes. As previously described, such probes can be of a variety of different
types.
IV. Pharmaceutical Compositions, Including Pharmaceutical
Compositions Adapted to be Preferentially Effective in Patients Having
Particular Genetic Characteristics
1. General
The methods of the present invention, in many cases ~wilI utilize
conventional pharmaceutical compositions, but will allow more
advantageous and beneficial use of those compositions due to the ability to
identify patients who are likely to benefit from a particular treatment or to
identify patients for whom a particular treatment is less likely to be
effective
or for whom a particular treatment is likely to produce undesirable or
intolerable effects. However, in some cases, it is advantageous to utilize
compositions which are adapted to be preferentially effective in patients who
possess particular genetic characteristics, i.e., in whom a particular
variance
or variances in one or more genes is present or absent (depending on whether
the presence or the absence of the variance or variances in a patient is
correlated with an increased expectation of beneficial response). Thus, for
example, the presence of a particular variance or variances may indicate that
a patient can beneficially receive a significantly higher dosage of a drug
than
CA 02335649 2001-O1-19
94
WO 00/04194 PCT/US99/1~440
a patient having a different variance or variances.
2. Regulatory Indications and Restrictions
The sale and use of drugs and the use of other treatment methods usually are
subject to certain restrictions by a govenmment regulatory agency charged with
ensuring the safety and efficacy of drugs and treatment methods for medical
use, and
approval is based on particular indications. In the present invention it is
found that
variability in patient response or patient tolerance of a drug or other
treatment often
correlates with the presence or absence of particular variances in particular
genes.
l0 Thus, it is expected that such a regulatory agency may indicate that the
approved
indications for use of a drug with a variance-related variable response or
toleration
include use only in patients in whom the drug will be effective, and/or for
whom the
administration of the drug will not have intolerable deleterious effects, such
as
excessive toxicity or unacceptable side-effects. Conversely, the drug may be
given
for an indication that it may be used in the treatment of a particular disease
or
condition where the patient has at least one copy of a particular variance,
variances,
or variant fonm of a gene. Even if the approved indications are not narrowed
to such
groups, the regulatory agency may suggest use limited to particular groups or
excluding particular groups or may state advantages of use or exclusion of
such
2o groups or may state a warming on the use of the drug in certain groups.
Consistent
with such suggestions and indications, such an agency may suggest or recommend
the use of a diagnostic test to identify the presence or absence of the
relevant
variances in the prospective patient. Such diagnostic methods are described in
this
description. Generally, such regulatory suggestion or indication is provided
in a
product insert or label, and is generally reproduced in references such as the
Physician's Desk Reference (PDR). Thus, this invention also includes drugs or
pharmaceutical compositions which carry such a suggestion or statement of
indication or warning or suggestion for a diagnostic test, and which may also
be
packaged with an insert or label stating the suggestion or indication or
warning or
3o suggestion for a diagnostic test.
In accord with the possible variable treatment responses, an indication or
suggestion can specify that a patient be heterozygous, or alternatively,
homozygous
for a particular variance or variances or variant form of a gene.
Alternatively, an
indication or suggestion may specify that a patient have no more than one
copy, or
zero copies, of a particular variance, variances, or variant form of a gene.
A regulatory indication or suggestion may concern the variances or variant
forms of a gene in normal cells of a patient and/or in cells involved in the
disease or
condition. For example, in the case of a cancer treatment, the response of the
cancer
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
cells can depend on the form of a gene remaining in cancer cells following
loss of
heterozygosity affecting that gene. Thus, even though normal cells of the
patient
may contain a form of the gene which correlates with effective treatment
response,
the absence of that form in cancer cells will mean that the treatment would be
less
s likely to be effective in that patient than in another patient who retained
in cancer
cells the form of the gene which correlated with effective treatment response.
Those
skilled in the art will understand whether the variances or gene forms in
normal or
disease cells are most indicative of the expected treatment response, and will
generally utilize a diagnostic test with respect to the appropriate cells.
Such a cell
to type indication or suggestion may also be contained in a regulatory
statement, e.g.,
on a label or in a product insert.
3. Preparation and Administration of Drugs and Pharmaceutical
Compositions Including Pharmaceutical Compositions Adapted to be
Preferentially
15 Effective in Patients Having Particular Genetic Characteristics
A particular compound useful in this invention can be administered to a
patient either by itself, or in pharmaceutical compositions where it is mixed
with
suitable carriers or excipient(s). In treating a patient exhibiting a disorder
of interest,
a therapeutically effective amount of a agent or agents such as these is
administered.
2o A therapeutically effective dose refers to that amount of the compound that
results in
amelioration of one or more symptoms or a prolongation of survival in a
patient.
Toxicity and therapeutic efficacy of such compounds can be determined by
standard pharmaceutical procedures in cell cultures or experimental animals,
e.g., for
determining the LDs° (the dose lethal to 50% of the population) and the
EDs° (the
25 dose therapeutically effective in 50% of the population). The dose ratio
between
toxic and therapeutic effects is the therapeutic index and it can be expressed
as the
ratio LDs°/EDs°. Compounds which exhibit large therapeutic
indices are preferred.
The data obtained from these cell culture assays and animal studies can be
used in
formulating a range of dosage for use in human. The dosage of such compounds
lies
3o preferably within a range of circulating concentrations that include the
EDs° with
little or no toxicity. The dosage may vary within this range depending upon
the
dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the therapeutically
effective dose can be estimated initially from cell culture assays. For
example, a
35 dose can be formulated in animal models to achieve a circulating plasma
concentration range that includes the ICs° as determined in cell
culture. Such
information can be used to more accurately determine useful doses in humans.
Levels in plasma may be measured, for example, by HPLC.
CA 02335649 2001-O1-19
96
WO 00/04194 PCTNS99/16440
The exact formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition. (See e.g. Fingl
et. al., in
The Pharmacological Basis of Thera eutics 1975, Ch. 1 p.l). It should be noted
that the attending physician would know how to and when to terminate,
interrupt, or
adjust administration due to toxicity, or to organ dysfunctions. Conversely,
the
attending physician would also know to adjust treatment to higher levels if
the
clinical response were not adequate (precluding toxicity). The magnitude of an
administrated dose in the management of disorder of interest will vary with
the
severity of the condition to be treated and the route of administration. The
severity
to of the condition may, for example, be evaluated, in part, by standard
prognostic
evaluation methods. Further, the dose and perhaps dose frequency, will also
vary
according to the age, body weight, and response of the individual patient. A
program comparable to that discussed above may be used in veterinary medicine.
Depending on the specific conditions being treated, such agents may be
formulated and administered systemically or locally. Techniques for
formulation
and administration may be found in Reminaton's Pha~rnaceutical Sciences, 18th
ed.,
Mack Publishing Co., Easton, PA (1990). Suitable routes may include oral,
rectal,
transdermal, vaginal, transmucosal, or intestinal administration; parenteral
delivery,
including intramuscular, subcutaneous, intramedullary injections, as well as
2o intrathecal, direct intraventricular, intravenous, intraperitoneal,
intranasal, or
intraocular injections, just to name a few.
For injection, the agents of the invention may be formulated in aqueous
solutions, preferably in physiologically compatible buffers such as Hanks's
solution,
Ringer's solution, or physiological saline buffer. For such transmucosal
2s administration, penetrants appropriate to the barrier to be permeated are
used in the
formulation. Such penetrants are generally known in the art.
Use of pharmaceutically acceptable carriers to formulate the compounds
herein disclosed for the practice of the invention into dosages suitable for
systemic
administration is within the scope of the invention. With proper choice of
carrier
30 and suitable manufacturing practice, the compositions of the present
invention, in
particular, those formulated as solutions, may be administered parenterally,
such as
by intravenous injection. The compounds can be formulated readily using
pharmaceutically acceptable carriers well known in the art into dosages
suitable for
oral administration. Such carriers enable the compounds of the invention to be
35 formulated as tablets, pills, capsules, liquids, gels, syrups, slurries,
suspensions and
the like, for oral ingestion by a patient to be treated.
Agents intended to be administered intracellularly may be administered using
techniques well known to those of ordinary skill in the art. For example, such
agents
CA 02335649 2001-O1-19
97
WO 00/04194 PCTJUS99/16440
may be encapsulated into liposomes, then administered as described above.
Liposomes are spherical lipid bilayers with aqueous interiors. All molecules
present
in an aqueous solution at the time of liposome formation are incorporated into
the
aqueous interior. The liposomal contents are both protected from the external
microenvironment and, because liposomes fuse with cell membranes, are
efficiently
delivered into the cell cytoplasm. Additionally, due to their hydrophobicity,
small
organic molecules may be directly administered intracellularly.
Pharmaceutical compositions suitable for use in the present invention include
compositions wherein the active ingredients are contained in an effective
amount to
to achieve its intended purpose. Determination of the effective amounts is
well within
the capability of those skilled in the art, especially in light of the
detailed disclosure
piovided herein. In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically acceptable carriers
comprising
excipients and auxiliaries which facilitate processing of the active compounds
into
preparations which can be used pharmaceutically. The preparations formulated
for
oral administration may be in the form of tablets, dragees, capsules, or
solutions.
The pharmaceutical compositions of the present invention may be manufactured
in a
manner that is itself known, e.g., by means of conventional mixing,
dissolving,
granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping
or
lyophilizing processes.
Pharmaceutical formulations for parenteral administration include aqueous
solutions of the active compounds in water-soluble form. Additionally,
suspensions
of the active compounds may be prepared as appropriate oily injection
suspensions.
Suitable lipophilic solvents or vehicles include fatty oils such as sesame
oil, or
synthetic fatty acid esters, such as ethyl oleate or triglycerides, or
liposomes.
Aqueous injection suspensions may contain substances which increase the
viscosity
of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or
dextran.
Optionally, the suspension may also contain suitable stabilizers or agents
which
increase the solubility of the compounds to allow for the preparation of
highly
3o concentrated solutions.
Pharmaceutical preparations for oral use can be obtained by combining the
active compounds with solid excipient, optionally grinding a resulting
mixture, and
processing the mixture of granules, after adding suitable auxiliaries, if
desired, to
obtain tablets or dragee cores. Suitable excipients are, in particular,
fillers such as
sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose
preparations such
as, for example, maize starch, wheat starch, rice starch, potato starch,
gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,
CA 02335649 2001-O1-19
98
WO 00/04194 PCT/US99/16440
disintegrating agents may be added, such as the cross-linked polyvinyl
pyrrolidone,
agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores
are
provided with suitable coatings. For this purpose, concentrated sugar
solutions may
be used, which may optionally contain gum arable, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and
suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be
added
to the tablets or dragee coatings for identification or to characterize
different
combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit
to capsules made of gelatin, as well as soft, sealed capsules made of gelatin
and a
plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain
the active
ingredients in admixture with filler such as lactose, binders such as
starches, and/or
lubricants such as talc or magnesium stearate and, optionally, stabilizers. In
soft
capsules, the active compounds may be dissolved or suspended in suitable
liquids,
such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In
addition,
stabilizers may be added.
EXAMPLES
Example 1 Gene Identification
Metabolic Pathways that Affect S-FUlFA Action
The biochemical pathways of 5-FU metabolism have been studied
extensively. Likewise, folate metabolism has been well investigated and the
enzymes that form and consume 5, 10-methylenetetrahydrofolate are well known.
The principal metabolic pathways that influence the pharmacologic action of 5-
FU
are summarized below.
De novo and salvage routes of pyrimidine nucleotide formation (S-FU
anabolism) and inhibition of thymidylate synthase
5-FU is a biologically inactive pyrimidine analog which must be
phosphorylated and ribosylated to the nucleoside analog fluorodeoxyuridine
monophosphate (FdUMP) to have clinical activity. FdUMP formation can occur via
several routes, summarized in Figure 1. 5-FU may be converted by uridine
phosphorylase to fluorouridine (FUdR; the reverse reaction is catalyzed by
uridine
nucleosidase) and then to fluorouridine monophosphate (FUMP) by uridine
kinase,
or FUMP may be formed from 5-FU in one step via transfer of a phosphoribosyl
group from 5-phosphoribosyl-1-pyrophosphate (PRPP), catalyzed by orotate
phosphoribosyl transferase. FUMP can be converted to FUDP and subsequently
FUTP by a nucleoside monophosphate kinase and nucleoside diphosphate kinase,
respectively. FUTP is incorporated into RNA by RNA polymerases, which may
CA 02335649 2001-O1-19
99
WO 00/04194 PCT/US99/16440
account in part for 5-FU toxicity as a result of effects on processing or
function (e.g.
translation). Alternatively, FUDP may be reduced to the dinucieotide level,
FdUDP
(fluorodeoxyuridine diphosphate) by ribonucleotide diphosphate reductase, a
heterodimeric enzyme. FdUDP can then be converted to FdUTP by nucleoside
diphosphate kinase and incorporated into DNA by DNA polymerases which may
account for some 5-FU toxicity. Fluoropyrimidine modified DNA may also be
targeted by the nucleotide excision repair process. The more important path of
FdUDP metabolism with respect to anticancer effects, however, is believed to
be
conversion to FdUMP by nucleoside diphosphatase (or cytidylate kinase, a
to bidirectional enzyme). dUMP is the precursor of dTMP in de novo pyrimidine
biosynthesis, a reaction catalyzed by thymidylate synthase and which consumes
5,10-methyienetetrahydrofolate, producing 7,8 dihydrofolate. FdUMP, however,
forms an inhibitory (probably covalent) complex with thymidylate synthase in
the
presence of 5,10-methylenetetrahydrofolate, thereby blocking formation of
thymidylate (other than by the saivage pathway via thymidine kinase). The
complex
anabolism of FdUMP can be simplified by giving the deoxyribonucleoside of 5-
FU,
5-fluorodeoxyuridine (also called floxuridine; FUdR), which can be converted
to
FdUMP in one step by thymidine kinase. However, FUdR is also rapidly converted
back to 5-FU by the bidirectional enzyme thymidine phophorylase.
S-FU catabolism.
Metabolic elimination of 5-FU occurs via a three step pathway leading to ~i-
alanine. The first and rate limiting enzyme in the elimination pathway is
dihydropyrimidine dehydrogenase (DPD), which transforms more than 80% of a
dose of 5-FU to the inactive dihydmfluorouracil form. Subsequently
dihydropyrimidinase catalyzes opening of the pyrimidine ring to form 5-fluoro-
~-
ureidopropionate and then (3-ureidopropionase (also called (3-alanine
synthase)
catalyzes formation of 2-fluoro-p-alanine. The first two reactions are
reversible.
The distribution of activity of these enzymes in human populations has not
3o been established, however, a recent population survey of urinary pyrimidine
levels in
1,133 adults revealed that levels of dihydrouracii range from 0 - 59 uM/g of
creatinine, while uracil levels ranged from 0 - 130 uM/g creatinine (Hayashi
et al.,
1996), suggesting variation in the activity of enzymes of pyrimidine
metabolism. It
is worth noting that in animal studies catabolites of 5-FU apparently account
for
some fraction of 5-FU toxicity (Davis et al., 1994; Spector et al., 1995).
This result
is the rationale for current human trials of 5-FU combined with DPD
inhibitors: if
the 5-fluoro- metabolites are responsible for toxicity, then blocking their
formation
by inhibition of DPD, while simultaneously decreasing 5-FU dosage to
compensate
CA 02335649 2001-O1-19
100
WO 00/04194 PCT/US99/16440
for the block in catabolism and excretion, should result in a better
therapeutic index.
Folinic acid conversion to tetrahydrofolate.
The conversion of FA to S,IOMTHF can occur via several routes, illustrated
in Figure 2.
Intracellular reduced folate levels can potentiate 5-FU action by increasing
5,10-methylenetetrahydrofolate levels (5,10-methyleneTHF; see center of Figure
2),
thereby stabilizing the ternary inhibitory complex formed with thymidylate
synthase
and FdUMP. This is the basis for therapeutic modulation of 5-FU with FA. As
can
to be seen in Figure 2, conversion of folinic acid (5-formylTHF) to 5,10-
methenylTHF,
the precursor of 5,10-methyleneTHF, requires methenyltetrahydrofolate
synthetase
(enzyme 2 in the Figure). Also, levels of 5,10-methyleneTHF may be affected
directly by the activity of methyleneltetrahydrofolate dehydrogenase,
methyleneltetrahydrofolate reductase, serine transhydroxymethylase and the
glycine
15 cleavage system enzymes (7, 8, 10 and 11 in Fig. 2), and indirectly by the
other
enzymes shown in the Figure.
Cell uptake ofpyrimidine nucleosides and folinic acid
Human cells have five concentrative nucleoside transporters with varying
2o patterns of tissue distribution (see review by Wang et al., 1997). Two
transporters,
one with preference for purines and one for pyrimidines have been cloned
recently
(Felipe et al., 1998). 5-FU entry into cells may be modulated by activity of
these
transporters, particularly the pyrimidine transporter, although one
prospective
randomized clinical trial in which the nucleoside transport inhibitor
dipyramidole
25 was paired with 5-FU and FA failed to show a difference in outcome compared
to 5-
FU/FA alone (Kohne et al., 1995). Several folate transport systems have been
identified in human cells. Folate receptor I (FRl) is a high affinity
(nanomolar
range) receptor for reduced folates. Three restriction fragment length
polymorphisms
(RFLPs) have been reported at the FRI locus (Campbell et al., 1991). Reduced
3o folates are also transported by folate receptor gamma and by a low affinity
(1 uM)
folate transporter. 15-fold variation in levels of folate transporter have
been
described in unselected tumor cell lines (Moscow et al., 1997).
Catalog allelic variation in enrymes that affect S-FU and FA action
35 Select genes for analysis of sequence variation
In accord with the pathway description above, variation in either expression
levels or intrinsic activity of the proteins involved in (i) cellular uptake
of
pyrimidines or reduced folate, (ii) conversion of 5-FU to the nucleotide form
FdUMP, FUTP or FdUTP, (iii) catabolism of 5-FU, (iv) conversion of folinic
acid to
CA 02335649 2001-O1-19
lUl
WO 00/04194 PCTNS99/16440
5,10-methylenetetrahydrofolate or (iv) depletion of cellular 5,10-
methylenetetrahydrofolate may be causally related to variation in clinical
effect of 5-
FU/FA. Table 3 below lists exemplary genes that will be, or already have been
screened for polymorphism.
Table 2
Conversion of
Folate Transport5-FU Anabolism5-FU CatabolismFoliaic Acid
to
5.10.MetbvIcaeTHF
Folate receptorUridine phosphorylaseDihydropyrimidineMethylenetetrahydrofolate
1 (a)
GenBank M28099 GenBank X90858Dehydrogenase synthase
GenBank 009178GenBank L38298
Folate receptorThymidine phosphorylase_ Methenyitetrahydrofolate
(J3) Dihydropyrimidinase
GenBank 102876 GenBank S72487GenBank D78011cyclohydrolase;
formylte-
trahydrofolate
synthetase;
Methenyltetrahydrofolate
dehydrogenase(onelocus)
GenBankJ04031
Folate TransporterOrotate phosphoribosyl-
Methylenetetrahydrojolate
(SLC19A 1 ) transferase Iabibitioa reductase
of dTMP
GenBank 019720 GenBankJ03626 Svathesis GenBank 009806
Folate receptorUridine KinaseThymidylate Serine transhydroxynteth-
(y) synthase
GenBank 232564 GenBank D78335GenBank X02308ylase 1 GenHank
L11931
Thymidine kinase Methionine synthetase
I
GenBank K02581; GenBank 050929
Thymidine Kinase
2
GenBank 077088
Ribonucleoside Glycine cleavage
reduct- system,
ase: MI su6unit Protein M:GenBank
GenBank M69175;
X59543 Protein P: GenBank
M64590;
Pyrimidiae TransportM2 subunit Folate Protein T: GenBank
D1381 I
GcnBank X59618Polv lutamation
Nucleoside transporterNucleoside FolylpolyglutamateDihydrofolate
1 diphosphate reductase
kinase, A subunitsynthetase GenBank 100140
GenBank 029200GenBank M98045
B subunit Folylpolyglutamate
GenBank X58965hydrolase
GenBank
There are 27 genes in the above Table. Six genes which have already been
surveyed for polymorphism are italicized. The following genes do not appear in
the
Table because there is no human cDNA in GenBank: 5-FU anabolism: Uridine
monophosphate kinase; 5-FU catabolism: b-ureidopropionase; Folate metabolism:
Glutamate formiminotransferase, Formiminotetrahydrofolate cyclodeaminase,
Formyltetrahydrofolate hydrolase, Formyltetrahydrofolate dehydrogenase, and
Protein L of the glycine cleavage system. Other genes not listed in the Table
include
DNA and RNA polymerises and DNA repair enzymes, some of which (e.g. DNA
polymerise b and RNA polymerise II 220 and 33 kD subunits) have already been
screened for polymorphism. Those additional genes are also useful in the
present
invention.
CA 02335649 2001-O1-19
102
WO 00/04194 PCT/US99/16440
For several potential candidate genes there are mammalian cDNAs in
GenBank but no human cDNA. For example, there is a 1,420. nucleotide full
length
rat (3-ureidopropionase cDNA. Four overlapping human ESTs (F06711, H19181,
811806 and W55897) span 691 nucleotides of the rat coding sequence with >90%
nucleotide identity. For selected candidate genes of likely importance, such
as p-
ureidopropionase, polymorphism analysis will be carried out on the available
human
sequence from dbEST.
to Example 2 Variance Identification - Variances in Genes That Can Affect 5-
FU/FA Action
Exemplary genes related to modulation of the action of 5-FU/FA have been
analyzed for genetic variation; thymidylate synthase, ribonucleotide reductase
(Ml
subunit only), dihydrofolate reductase and dihydropyrimidine dehydrogenase
cDNAs. 36 unrelated individuals were screened using 6 SSCP conditions and DNA
sequencing. Other investigators have identified variances in MTHFR, methionine
synthase and folate receptor. These findings are summarized in Table 3.
Table 3: Variation in Genes Which Modulate 5-FUIFA PharmacnlnQv
Geae Name Variances Heterory-
(Gcnbank gote Comments
accession Ba:e RNA ProteinFr uencv
no.)
Cytidine 79 T or G ys27glu> 40%
Aesminase
L27943
Dihydrofolate721 T or A 20%
Reductase 829 C or T 14%
(J00140) Rsal RFLP 23, 33, 3 alleles
43%
SCTF I 26%
Rsal RFLP 32% uniue Rsal
RFLP
Dihydropyrimidinase1001 A or G In334argrare AIt found in
patients
(D7801 I 1303 G or A Iy435arg with DHP deficiency
)
203 G or C r68arg
1468 G or C g490thr
1078 T or C 360arg rare
812 to Insertionrcmat.
814 A term.
CA 02335649 2001-O1-19
103
WO 00/04194 PCT/US99/1b440
Dihydropyrimidine166 T or C cys29argI 1
Dehydrogenase577 A or G et166va19%
(U09178) 3925 A or G ' UTR 35%
3937 T or C ' UTR 38%
3432 T of C ' UTR 10%
g2lgln rare
a13351eurare
638 A or G 186cys 2%
784 C or T g235trprare
296 to Delete rerttat.rare
299 TCAT term.
1682 G of A 534asn 0.5-3%
1708 A or G Ie543va17-35%
'
exoNintronG or A el. 1% 73% in DPD
581-635 deficiency
14 delete remat. rare
C terttt.
I897 G or A a1732i1e1-7%
2275 G or A g886hisrare
2738 A or T 974trp rare
3002 G or T a1995pherare
2983
Folate Receptor One Msp I and
a 2 Pst I
M28099 RFLPs
Folate receptor330-331 2 by deletionrecnat.75%
Y Term.
232564
Foiate Transporter341 C or G llent 1
(SLC 19A
1 )
U 19720
Folylpoiyglutamate1747 G or T ' UTR 2%
Synthetase 1900 T oc C ' UTR 50%
M98045
Glycine cleavage710 C or G ' UTR 7%
System: protein
H
M69175
Glycine cleavage 564i1e rare 70% in NKH
patiatts
System: protein
P
M64590
Glycine cleavage277 G or T aISOleu2%
System: protein1073 G or A g3151ys1
T
(D13811 ) 1083 G or A ilent 2%
1773 C or T 'UTR 3%
3
Methenyltetrahydro-454 G or A g1341ys22%
folate cyclohydrolase969 C or G 1n306g1u!
1614 C of T ilent 1
2011 G or A rg653g1n35%
r 293hisrare
Methylenetetra-129 C or T Low Both the amino
acid
hydrofolate 677 C or T 1a223va148% changes affect
MTHFR
Reductase 1068 C or T low activity.
(U09806) 1298 C or A 1a430g1uhigh
308 T or C ilent 5-39%
rare Rare mutations
found in
MTHFR deficienc
Methionine 2756 G or A sp919g1y19-29% Affects folate
levels in
Synthase 3970 T or C ilent colon cancer
S patients.
(U50929, I 158 G or A ys225tryrare
U73338))
1004 G or T la to
ser
rare Rare mutations
Found in
MS deficienc
Nucleoside gl1
RFLP
Diphosphate
kinase B
X58965
CA 02335649 2001-O1-19
104
WO 00/04194 PCT/US99/16440
Ribonucleotide1037 C or A 33%
Reductase, 2410 A or G 40%
M 1
(X59543) 2419 A or G 20%
2717 T Or A 19%
2724 T in/del 19%
aCI RFLP47%
Ribonucieotide524 C or G ilent 1
Reducfase, 1636 C or T ' UTR 1
M2
X59618 2259 T or C ' UTR 1
Serine Hydroxy-1444 u474phe 23%
methyltransferaseI 541 C or T ' UTR 26%
(cytolic)
L11931)
Thymidine 90 T or C ilent 50%
kinase I
(K02581 ) 279 G or A ilent 13%
282 G or A ilent 30%
772 G or A ' UTR 26%
867 G or A ' UTR 50%
acl RFLP40%
stEll 2 34 3 alleles .
RFLP 64 %
Thymidine 1480 T or C ' UTR 9%
kinase 2
U77088
Thymidine 601 G or C ' UTR 3%
Phosphorylase3673 A or G
(PD-ECGF) 3576 T or C ilent 54%
(572487) tare Rare mutations
found in
MNGIE atients
Thymidylate 276 T or C 33his rare
Synthase 1140 C or T 53%
1210 A or G 42%
(X02308) 1571 A or T 53%
28-34 ' re double:
nt ats . R 19%
ion
Uridine mono.742 G or C 1y213a1a23%
Phosphate 1575 A or G ' UTR 1
synthetase
(103626) rare Raro mutations
found in
OroNc aciduria
atients
A more complete catalog of genetic variances is shown in the following table
for the dihydropyrimidine dehydrogenase (DPD) gene.
Table 4
Variances in Dihydropyrimidine Dehydrogenase Geae
Valiant Variant Variant Effect
base base o0
1
uucleodde (frequency)l mRNA & Comments
codou fre uencvrotein
Arg allele has no
activity when
166 (29) T (6?!70)C (8/70)cys29arg exprossed in E.
Coli (Vreken,
Human Genetics.
1997
577 ( 166)A (69/72)G (3/72)met 166va1Located in highly
conserved
domain; no functional
studies
Ttp allele has no
activity when
784 (235) C T arg235trpexpressed in E.
Coli (Vrcken,
Human Genetics.
199?
1682 (534)G ( 148/150)A (2/150)ser534asnApparently little
or no functional
effect in anent
cells.
1708 (543)A (34/46)G ( i i1e543va1Apparently little
2146) or no functional
effect in anent
cells.
55 missing amino
acids result in
intron G A no exon unstable protein.
13 14 Mutant allele
(destroys may be present in
5' GT -.I % of Flnns;
s lice very rare in other
site arou s, but
CA 02335649 2001-O1-19
1~5
WO 00/04194 PCT/US99/1b440
immediately detected in 8 of
i 1 patirnts with
after com lete deficirnc
nt 1986 .
1897 (606)- deletion ftameshiftLow/no activity
of C allele; reported
in
oni one atient so
far.
His allele has -25%
of normal
2738 (886)G A arg886hisactivity when expressed
in Coti
Vrekrn. Human Genetics,
'97
Val allele apparently
has very low
3002 (974)A T asp974va1or no activity in
patient sample.
Very low frequency
allele (<0.2%
in Americans .
3925 A (41/62)G (21/62)3' U'TR Two high frequrncy
variances, 12
nt apart but not
in complete
_ C (40/64)T (24/64)3' tJTR linkage disequilibrium.
3937 J ~
Variances in the exemplary genes above which affect the activity of the
corresponding gene product have the potential to modulate the activity of 5-
FU/FA
and thereby provide predictive capability concerning the efficacy of such
treatment
in a particular patient. As discussed above, such predictive capability can
further be
provided by the joint determination of multiple variances, in one or a
plurality of
genes or both. Similarly, such variances can provide such predictive
capability for
other treatments, e.g., treatments with other compounds, which involve these
genes.
t0
Example 3 Relationship of Genes to Drug Response - 5-flurouracil
5-fluorouraciI (5-FU) is a widely used chemotherapy drug. The effectiveness
of 5-FU is potentiated by folinic acid (FA; generic name: leukovorin). The
t5 combination of 5-FU and FA is standard therapy for stage III/IV colon
cancer.
Patient responses to 5-FU and S-FU/FA vary widely, ranging from complete
remission of cancer to severe toxicity.
Clinical Use and E, fjectiveness of S-FU and S-FUIFA
20 5-FU is a pyrimidine analog in clinical use since 1957. 5-FU is used in the
standard treatment of gastrointestinal, breast and head and neck cancers.
Clinical
trials have also shown responses in cancer of the bladder, ovary, cervix,
prostate and
pancreas. The remainder of this discussion will concern colorectal cancer. S-
FU is
used both in the adjuvant therapy of Dukes Stage B and C cancer and in the
25 treatment of disseminated cancer. 5-FU alone produces partial remissions in
10-
30% of advanced colorectal cancers, however only a few percent of patients
have
complete remissions, and no benefit in survival has been demonstrated.
In the last 15 years a variety of biochemically motivated strategies for
modulating 5-FU activity have been tested. For example, 5-FU has been used in
3o combination with PALA, a pyrimidine synthesis inhibitor, to deplete
cellular pools
CA 02335649 2001-O1-19
106
WO 00/04194 PCTNS99/16440
of UTP and thereby enhance formation of FUTP; in combination with
methotrexate,
to inhibit purine anabolism, leading to increased PRPP levels and consequent
increased conversion of 5-FU to its active nucleotide metabolites; and in
combination with folinic acid, which increases intracellular pools of reduced
folate,
s driving formation of the ternary inhibitory complex formed by 5,10
methylenetetrahydrofolate, FdUMP and thymidylate synthase. Levamisole,
interferon and alkylating agents have also been used in combination with 5-FU.
5-
FU/Levamisole and 5-FU/FA are widely used in the adjuvant treatment of colon
cancer, while 5-FU/FA is the most commonly used regimen for advanced
colorectal
to cancer. Six of seven prospective randomized trials of 5-FU/FA vs. 5-FU
alone in
patients with advanced cancer have demonstrated up to two fold higher response
rates to 5-FU/FA, while two of the studies also showed increased survival.
Two major dosing regimens are used: 5-FU plus low dose FA given for five
consecutive days followed by a 23 day interval, or once weekly bolus iv 5-FU
plus
15 high dose FA. The higher FA dose results in plasma FA concentrations of 1
to i 0
uM, comparable to those required for optimal 5-FU/FA synergy in tissue
culture,
however low dose FA (20 mg/m2 vs. 500 mg/m~) has produced comparable clinical
benefit. Ongoing clinical trials are designed to further test new drug
combinations.
In summary, relatively few patients - in the single digits - live longer as a
result of 5-
2o FU/FA, although significantly more have partial disease remission. The
factors that
determine which patients respond or have side effects are not known.
S-FU modulators
Leukovorin (folinic acid) is the most widely used S-FU modulator, however
25 a variety of other molecules have been used with 5-FU, including, for
example,
interferon-alpha, hydroxyurea, N-phosphonacetyl-L-aspartate, dipyridamole,
levamisole, methotrexate, trimetrexate glucuronate, cisplatin and
radiotherapy. S-1 is
a novel oral anticancer drug, composed of the 5-FU prodrug tegafur plus
gimestat
(CDHP) and otastat potassium (Oxo) in a molar ratio of 1:0.4:1, with CDHP
3o inhibiting dihydropyrimidine dehydrogenase in order to prolong 5-FU
concentrations in blood and tumour and Oxo present as a gastrointestinal
protectant.
Some of these regimens show promising results, but no clear improvement over 5-
FU/leukovorin. The clinical development and use of regimens containing 5-FU
plus
modulators may be facilitated by the methods of this invention.
Toxicity of S-FU and Folinic Acid
5-FU toxicity has been well documented in randomized clinical trials.
CA 02335649 2001-O1-19
1~7
WO 00/04194 PCT/US99/16440
Patients receiving 5-FU/FA are at even greater risk of toxic reactions and
must be
monitored carefully during therapy. A variety of side effects have been
observed,
affecting the gastrointestinal tract, bone marrow, heart and CNS. The most
common
toxic reactions are nausea and anorexia, which can be followed by life
threatening
mucositis, enteritis and diarrhea. Leukopenia is also a problem in some
patients,
particularly with the weekly dosage regimen. In a recent randomized trial of
weekly
vs. monthly 5-FU/FA, there were 7 deaths related to drug toxicity among 372
treated
patients (1.9%; Buroker et al. 1994). 31 % of patients receiving the weekly
regimen
suffered diarrhea requiring hospitalization for a median of 10 days. Other
severe
to toxicities, which occured at lower frequency, included leukopenia and
stomatitis. In
another example, 36% of patients receiving weekly bolus 5-FU plus FA (500
mg/m2), in a NSABP trial suffered NCI grade 3 toxicity (Wolmark et al., 1996).
Clearly, toxicity is a major cost of 5-FU/FA therapy, measured both in patient
suffering and in financial terms (the cost of care for drug induced illness).
Other Factors
Many non-genetic factors can influence the response of cancers to drugs,
including tumor location, vasculature, cell growth fraction and various drug
resistance mechanisms. It is therefore not possible to explain all
heterogeneity in
2o response to 5-FU/FA administration by genetic variation. However, based on
genetic studies of other quantitative traits it appears that a significant
fraction of
variation in drug response is due to genetic variation.
Example 4 Genetic Component of Drug Response Variability
Genetically Determined Variation in Response to 5-FU: Studies of
Dihydropyrimidine Dehydrogenase Deficiency
Dihydropyrimidine Dehydrogenase Deficiency is Associated with S-FU
3o Toxicity
S-FU is inactivated by the same metabolic pathway as thymine and uracil
(see above). DPD catalyzes the first, rate limiting step in pyrimidine
catabolism and
accounts for elimination of most 5-FU. Normal individuals eliminate S-FU with
a
half life of ~10-15 minutes and excrete only 10% of a dose unchanged in the
urine.
In contrast, people genetically deficient in DPD eliminate 5-FU with a half
life of
~2.5 hours and excrete 90% of a dose unchanged in the urine (Diasio et al.,
1988).
DPD deficiency has two clinical presentations: (i) an inborn error of
metabolism
causing some degree of neurologic dysfunction or (ii) asymtomatic until
revealed by
exposure to 5-FU or other pyrimidine analogs. With either presentation there
is
CA 02335649 2001-O1-19
t~$
WO 00/04194 PCT/US99/16440-
combined hyperuraciluria and hyperthyminuria. The vastly increased 5-FU half
life
in DPD deficient individuals causes severe toxicity and even death. Recently
several
mutations have been identified in DPD genes of deficient individuals (Wei et
al.,
1996), however none of these alleles appears to occur at appreciable
frequency, so
the cause of wide population variation in DPD levels is still not understood.
Dihydropyrimidine dehydrogenase (DPD) inhibitors
More than 85% of an injected dose of 5-FU is rapidly inactivated by
dihydropyrimidine dehydrogenase (DPD) to therapeutically inactive catabolic
products, however there is evidence that said catabolic products may be toxic
to
normal tissues. This has led to the development of DPD inhibitors with the aim
to
modify the therapeutic index of 5-FU. Several inhibitors in combination with 5-
FU
are under preclinical and clinical evaluation, including uracil .and 5-chloro-
2,4-
dihydroxy pyridine, as modulators of 5-FU derived from its prodrug tegafur and
5-
15 ethynyluracil as a modulator of 5-FU itself (Eniluracil, 776C85; Glaxo
Wellcome
Inc, Research Triangle Park, NC). Other compounds with DPD inhibitory activity
include 5-propynyluracil. (For a review of DPD inhibitors see: Diasio, RB
Improving 5-FU with a Novel Dihydropyrimidine Dehydrogenase Inactivator,
Oncology 1998, Mar; 12(3 Suppl. 4):51-6.)
Population Studies of DPD Activity Show Wide Variation
Population surveys of DPD activity in normal individuals have been
performed using blood and liver samples. These studies reveal a broad unimodal
Gaussian distribution of DPD activity over a 7 to 14 fold range, with some
individuals having very low or even undetectable levels. For example Etienne
et aI.
(1994) report DPD activity ranging from .065 to .559 nM/min/mg protein in a
study
of 152 men and 33 women, while Fleming et al. (1993) found DPD activity in 66
cancer patients varied from .17 to .77 nM/min/mg protein. Lu et al (1995)
found 18-
fold variation in liver DPD assayed in 138 individuals. Milano and Etienne
(1994)
suggested that the frequency of heterozygous and homozygous deficiendy is 3%
and
.1 %, respectively. The DNA sequence alterations responsible for null DPD
alleles
do not account for the high population variability (Ridge et al., 1997).
DPD Levels Correlate with Response to S-FU
Intratumoral DPD levels have been measured in patients receiving 5-FU
chemotherapy. When complete responders were compared to partial or
nonresponders, DPD levels were lower in the compete responders (Etienne et
al.,
1995). Leukocyte DPD levels have also been measured in patients receiving 5-
FU/FA chemotherapy. When patients were divided into 3 groups: high, medium and
CA 02335649 2001-O1-19
109
WO 00/04194 PCT/US99/16440
low DPD activity, the frequency of serious side effects was highest in the low
DPD
group and vice versa (Katona et al., 1997).
Biochemical Studies of Alternate Allelic Forms of DPD
The power of genetic analysis can be augmented by biochemical studies of
alternate allelic forms of enzymes. Biochemical data on the distribution of
activity
of a series of enzymes in a biochemical pathway provides the basis for
metabolic
flux analysis (Keightly, 1996). It is beyond the scope of this proposal to
exhaustively analyze biochemical variation in the enzymes of pyrimidine and
folate
metabolism. However, since we have identified new variances in DPD that may
affect enzyme expression or activity, and because DPD is already proven to
play a
role in 5-FU response, we will determine the relationship between genotype and
biochemistry for this enzyme.
DPD cDNAs have been cloned from a variety of higher eukaryotes and
binding sites for its cofactors, prosthetic groups and substrate have been
defined
experimentally or by analogy with known consensus motifs (Yokata et al.,
1994).
The DPD polymorphisms that affect protein sequence occur at amino acids 29
(cys/arg) and 166 (met/val) in the amino-terminal one-third of the protein.
Phylogenetic comparison of this region from boar, human, cow, fly, and
bacteria
(see below) shows that there are actually two highly conserved motifs that
resemble
either iron/sulfur or zinc binding motifs, the latter being more likely due to
the
spacing of the cysteine residues. The region around the met/vaI polymorphism
at
amino acid 166 is highly conserved. Even the spacing of the putative zinc-
finger
domains is maintained between distantly related species, hinting at their
importance.
Since amino acid 166 is close to a highly conserved (and probably functionally
important) region and is itself conserved, being a methionine in all species,
it seems
likely that perturbations in this position would have consequence. The
polymorphism substitutes a long amino acid side chain capable of hydrogen
bonding
(methionine) for a compact, hydrophobic amino acid (valine). The region around
amino acid 29 is not as well conserved.
Common DPD Haplotypes
Eight haplotypes from 58 chromosomes (29 individuals) have been
identified. Using methods described above, the DNA from these samples were
analyzed by PCR. The single base pair substitutions at four locations were
identified
as allelic haplotypes, e.g. base pair number 166, 577, 3925, 3937. Base pair
positions, 3925 and 3937 are located in the 3 prime untranslated region of the
cDNA
and base pairs 166 and 577 are within the coding region.
CA 02335649 2001-O1-19
110
WO 00/04194 PCT/US99/16440
Table 5
Identified DPD Hanlotwes
Base
Position
No.
Chromosomes166 577 3925 3937
14 T A G C
24% c met
s
16 T A A C
28%) C met
S
16 T A A T
28% C met
s
4 C A A T
7% ar met
3 C A G C
5% 8r mCt
3 C A A C
5% 8f met
1 T G G C
2% C vat
S
I T G A C
2% c val
s
Total=58(100%)~-._
I
Example 5 - Exemplary Genes involved in Folate Transport and Metabolism
While examples above concern 5-FU/FA action and genes which are
expected to modulate such action, it is also useful to utilize genes involved
in folate
transport and metabolism generally. A number of these genes are also involved
in 5-
FU/FA action. Genes known to be involved in folate transport and metabolism
are
listed in the table below, along with available GenBank accession numbers for
deposited sequences.
Table 6
Gene Field: Folate Transport & Metabolism
Folate Folate PolyglutamationBiosynthesis,
Degradation
and Interconversioa
of
Trana orters Folates
Folate receptorFolylpolyglutamateFormiminotetrahy-Glutamate form-
I(a)
(GenBank M28099)synthetase drofolate iminotransferase
GenBank M98045c clodeaminase
Folate receptor Methenyltetrahy-Formyitetrahydrofolate
((3)
GenBank J02876 drofotate synthetaseh drolase
Folate receptor Mcthylcnetetrahy-Methylenetetrahydrofolate
(y)
(GenBank 232564) drofolatc dchydrogenasesynthase
GenBank L38298
Folate Transporter Methionine Methylenetetra6ydrojoJate
rynthetase
(SLC19A 1 ) GenBank 050929redueJtue
GenBank 019720 GenBank 009806
Dihydrojolate Serine transhydroxy-
reduetase
Folate Inhibition GenBank J00140methylase 1
of dTMP
Absorbtion Svntbesis GenBank L 1 t
931
CA 02335649 2001-O1-19
111
WO 00/04194 PCT/US99/16440
Ptaoyl-r-glutamylThymidylate Methenyltetrahy-Glycine cleavage
synthase rystem,
carboxypeptidaseGenBank X02308drofoiate cyclohy-Protein H: GenBank
M69175;
drolase; formylte-Protein P: GenBank
M64590;
trahydrofolateProtein T: GenBank
D13811;
synthetase; Protein L
Meth-
enyltetrahydrofol-ateFormyltetrahydrofolate
dehydrogenase dehydrogenase
(one
locus)
GenBankJ04031
Genes affecting the action of drugs which modulate folate metabolism.
There are 24 genes in the Table, four of which we have already surveyed for
polymorphism (italicized genes). The genes with GenBank numbers are currently
being screened for variances. Genes lacking GenBank numbers are not yet
represented in GenBank as full length cDNAs; but will be scanned using
relevant
EST collections or using sequences from other publicly available sources.
to
Example 6 - Drugs Targeting Genes Involved in Folate Transport and Metabolism
In concert with the identification of useful genes involved in folate
transport
and metabolism, the table below identifies certain drug classes used for
treatment of
identified disorders, along with a brief characterization of the action of the
drug.
15 Exemplary drugs are identified within the individual classes. Variable
response of
patients to administration of drugs of these classes, or administration of the
specific
drugs can be used in identifying variances responsible for such variable
response.
As described above, those variances can then be used in diagnostic tests,
methods of
selecting a treatment, methods of treating a patient, or other methods
utilizing
2o genetic variance information as otherwise described.
Table 7
Drug Field: Folate Transport & Metabolism
Disease/
Indication
Dru Class Mec6snism of Action E:em h Drn
s
Cancer Reduced folatesBlock dTMP biosynthesis leukovorin,
by inhib-icing L-leu-
thymidylate synthase (TS)kovorin, citrovor-um
via formation of
ternary complex invol-vingfactor (used
TS, 5- with 5-
fluorodeoxyuridine and fluorouracil
5,10- or related
meth lenetetrahvdrofolatedru s
Cancer Reduced folatesRescue bone marrow from leukovorin,
lethal toxicity after L-leu-
high dose methotrexate kovorin, citrovor-um
factor
Cancer Folate analogsBlock de novo purine biosynthesisMethotrexate,
by
(antifolates)inhibiting dihydrofolate aminopterin,
reduc-tale, TS, dide-
azatetrah
drofolate
ProliferativeFolate analogsBlock de novo purine biosynthesisMethotrexate,
skin by
~ ~ inhibiting dihydrofolate aminopterin,
diseases (antifolates)reduc-tale, TS, dide
(psoriasis) ~
azatetrah
drofolat
CA 02335649 2001-O1-19
112
WO 00/04194 PCT/US99/16440
lmmunosup- Folate analogsBlock de novo purine Methomxate,
biosynthesis by
pression (antifolates)inhibiting dihydrofolateaminopterin,
reduc-tale, TS, dide-
azatetrahvdrofolate
Autoimmune Folate analogsBlock de nowv purine Methotrcxate,
biosynthesis by
diseases, (antifolates)inhibiting dihydrofolatesminopterin,
such as reduc-case, TS, dide-
rheutnatoid azatetrah drofolate
arthritis
Folate deficiencyFolic acid Increase folates for Folic acid
purine and pyrimidine
bios thesis
CardiovascularFolic acid Reduce plasma homocysteineFolic acid
levels in
disease (prevent patients with low MTHFR
levels
atherosclerosis
Prevent spinsFolic acid Reduce plasma homocyateineFolic acid
bifida levels in
tients with low MTHFR
Levels
Table 7. Drugs which affect or are affected by folate metabolism. A wide
spectrum of diseases are treated with drugs that affect folate metabolism.
Some
drugs are used in the treatment of several diseases. All of the listed drugs
are
frequently used in combination with other drugs. For example methotrexate is
used
in cancer chemotherapy with cytoxan and fluoruracil to treat breast cancer,
among
other combinations.
Folate analogs
1o Many novel antifolate compounds with unique pharmacoiogic properties are
currently in clinical deveiopment. These newer antifolates differ from
methotrexate,
the most widely used and studied drug in this class, in terms of their
lipophilicity,
cellular transport mechanism, level of polyglutamation, and specificity for
inhibiting
folate-dependent enzymes, such as dihydrofolate reductase, thymidylate
synthase, or
15 glycinamide ribonucleotide formyltransferase. The clinical development and
use of
these new compounds can be affected by the methods of this invention. The new
folate analogs include quinazoline derivatives such as ZD1694 (Tomudex,
AstraZeneca) which requires Reduced Folate Carrier (RFC) mediated cell uptake
and polyglutamation by Folylpolyglutamate Synthetase (FPGS); ZD9331
20 (AstraZeneca), which requires the RFC but is not polyglutamated by FPGS;
LY231514 (Eli Lilly Research Labs, Indianapolis, III is a multitargeted
pyrrolopyrimidine analogue antifolate which requires the RFC and
polyglutamation;
GW1843 (1843U89, GlaxoWellcome) is a benzoquinazoline compound with potent
TS inhibitory activity, and which enters cells via the RFC but is
polyglutamated only
25 to the diglutamate, which leads to higher cellular retention without
augmenting TS
inhibitory activity; AG337 (p.o. and i.v. forms) and AG331 (both by Agouron,
La
Jolla, CA, now part of Warner Larnbert) are lipophilic TS inhibitors with
action
independent of the RFC and polyglutamation by FPGS; trimetrexate (US
Bioscience) is a ; Aminopterin is an older drug which has received renewed
attention
3o recently; edatrexate, piritrexim and lometrexol are other antifolate drugs.
More
CA 02335649 2001-O1-19
113
WO 00/04194 PCT/US99/16440
generally, 5,8-dideazaisofolic acid (IAHQ), 5,10-dideazatetrahydrofolic acid
(DDATHF), and 5-deazafolic acid are structures into which a variety of
modifications have been introduced in the pteridine/quinazoline ring, the C9-
N10
bridge, the benzoyl ring, and the glutamate side chain (see article below).
Also
Lilly have recently synthesized a new series of 2,4-diaminopyrido[2,3-
d]pyrimidine
based antifolates which are being evaluated both as antineoplastic and
antiarthritic
agents.
Other Therapeutic Categories in which Folate or Pyrimidine Pathwyas may be
to Relevant to Drug Development
1 ) Cardiovascular Drugs
Homocysteine is a proven risk factor for cardiovascular disease. One
important role of the folate cofactor 5-methyltetrahydrofolate is the
provision of a
methyl group for the remethylation of homocysteine to methionine by the enzyme
15 methionine synthase. Variation in the enzymes of folate metabolism, for
example
methionine syntase or methylenetetrahydrofolate reductase (MTHFR), may affect
the levels of 5-methyltetrahydrofolate or other folates that in turn influence
homocysteine levels. The contribution of elevated homocysteine to
atherosclerosis,
thromboembolic disease and other forms of vascular and heart disease may vary
20 from one patient to another. Such variation may be attributable, at least
in part, to
genetically determined variation in the levels or function of the enzymes of
folate
metabolism described in this application. Assistance of clinical development
or use
of drugs to treat said cardiovascular diseases might be afforded by an
understanding
of which patients are most likely to benefit. This is true whether the drugs
are aimed
25 at the modulation of folate levels (e.g. supplemental folate) or at other
known causes
of cardiovascular disease (e.g. lipid lowering drugs such as statins, or
antithrombotic
drugs such as salicylates; heparin or GPIIIa/IIb inhibitors). It may, for
example, be
desirable to exclude patients whose disease is significantly attributable to
elevated
homocysteine from treatment with agents aimed at the amelioration of other
3o etiological causes, such as elevated cholesterol. Thus, the understanding
of variation
in the enzymes of folate transport and metabolism may be important in
evaluating
drugs used to treat atherosclerosis, thromboembolic diseases and other forms
of
vascular and heart disease.
35 2) CNS drugs
The observation that phencyclidine, an NMDA receptor antagonist, induces a
psychotic state closely resembling schizophrenia in normal individuals has led
to
attempts to modulate NMDA receptor function in schizophrenic patients. The
amino
CA 02335649 2001-O1-19
114
WO 00/04194 PCT/US99/16440
acid glycine is an obligatory coagonist (with glutamate) at NMDA receptors
(via its
action at a strychnine-insensitive binding site on the NMDA receptor complex),
and
consequently glycine or glycinergic agents (e.g. glycine, the glycine receptor
partial
agonist, D-cycloserine, or the glycine prodrug milacemide) have been tried as
an
adjunct to conventional antipsychotics for the treatment of schizophrenia.
Several
trials have demonstrated a moderate improvement in negative symptoms of
schizophrenia. Because the folate pathway modulates levels of serine and
glycine,
the endogenous levels of glycine in neurons may affect the response to glycine
or
glycinergic drugs. In particular, interpatient variation in glycine metabolism
may
to affect drug efficacy.
Example 7 - Genes Related to Pyrimidine Transport and Metabolism
15 Similar to the genes involved in folate transport and metabolism, genes
involved in the related pathways of pyrimidine transport and metabolism are
useful
in the aspects of the present invention, e.g., for identifying variances
responsible for
variable treatment response, diagnostic methods, and methods of selecting a
patient
to receive a treatment. Exemplary genes are provided below and are further
2o identified by cellular function. Genes involved in those functions are
generally
useful in the present invention.
Table 8
Gene Field: Pyrimidine Transport & Metabolism
Pyrimidine Pyrimidine Pyrfmidine Catabolism
Transport Bfosyotbeafs
ds aovo
and
Salvo a Patbwa
:
Equilibrative Uridine phosphorylaseRibonucleosideDihydropyrtnridine
nucleoside
transporter GenBank X90858reductase: Dehydrogenase
1
M! subweit GenBank 009178
GenBank X59543
M2 subunit
GenBank X59618
Equilibrative Thymidine Nucleoside Dihydropyrimidinase
nucleoside
transporters phosphorylasediphosphate GenBank D7801
2, 3, 4 & kinase, I
5
GenBank 572487A subunit
GrnBank 029200
Concentrative Orotate (3-ureidopropionase
nucleoside
transporters phosphoribosyl-B subunit
transferase GenBank X58965
GenBank J03626
Uridine KinaseUridine mono-Cytidine deaminase
GenBank D78335hos hate
kinase
Thymidine DeoxycytidylatedCMP deaminase
kinase
GenBank K02581;kinase
Thymidine
Kinase 2
GenBank 077088
CA 02335649 2001-O1-19
115
WO 00/04194 PGTNS99/16440
Deoxycytidine p-alsnine-pyruvate
kinase
lnblbltion aminotransferase
of dTMP
S thesis
Tiiymidy(ate (3-alanint-a-detogiutarate
syntbase
GenBank X02308 aminotransferase
Table 8. Genes affecting the action of drugs which modulate pyrimidine
metabolism. We have already surveyed three of the above genes for polymorphism
(italicized genes). The genes with GenBank numbers are currently being
screened
for variances. Genes in the table lacking GenBank numbers are not yet
represented
in GenBank as full length cDNAs; but can be evaluated using relevant EST
collections. Genes not listed in the Table but related to the mechanism of
action of
pyrimidine analogs include DNA and RNA polymerises and subunits and DNA
to repair enzymes, some of which (e.g. DNA polymerise p and 220 kD and 33 kD
subunits of RNA polymerise II) have already been screened for polymorphism.
Such additional genes can also be used in the present invention.
15 Example 8 - Drugs Targeting Genes Involved in Pyrimidine Transport &
Metabolism
As was described above for drugs modulating genes involved in fotate
transport and metabolism, particular drug classes and exemplary drugs are
identified
in the table below which modulate the action of pyrimidine transport and
2o metabolism genes. These classes of drugs and exemplary drugs are similarly
useful
for identifying variances which affect the action
Table 9
Drug Field: Pyrimldine Transport & Metabolism
Disease) Dru Class Mechanism of Action Exem la Dru
lndicatioo s
Cancer FluoropyrimidinesBlock dTTP biosynthesis 5-FU, fluorode-
by inhib-icing
thymidylate synthase; oxyuridine,
inhibit replication, flu-
_ transcription and/or orodeoxyuridine
repair by incorporation
into DNA and RNA. monophosphate,
to afar ftorafur.
Cancer DihydropyrimidinePotentiate fluoropyrimidines5-ethynyluracil;
by blocking their
dehydrogenase catabolism, increasing 5-propynyluracil;
inhibitors half life. 2,6
dih drox -ridine
Cancer Cytidine analogsIncorporation into DNA Cylosino arabino-side,
and conse-quent
inhibition of DNA synthesisgemcitabine,
(replication, 5-
transcription, repair). azacytidine,
5-
azacytosine
ara-
binoside, others.
Cancer Other 'midine Inhibition of nucleic
analo s acid synthesis
Cancer Ribonucleotide Inhibit reduction of Hydroxyurea
reductase ribonucleotides (e.g.
CTP)
inhibitors to deoxvribonuc-leotides
(dCTP
CA 02335649 2001-O1-19
116
WO 00/04194 PCT/US99/16440
Cancer Nuclcotide/nucieosideBlock import of cytotoxicdipyridamole,
pyrimi-dine analogs BIBW 22
uptake inhibitors(protective effect), or (a di
block.import of nomad pyridamole
analog),
pyrimidine nucleotides, nitroben-rylthioinosine
thereby reducing sal-
vage synthesis and increasing
need for de
novo s thesis, includin
dTMP synthesis.
Table 9. Genes affecting the action of drugs which modulate pyrimidine
metabolism. A variety of proliferative diseases, especially cancer, are
treated with
drugs that affect pyrimidine metabolism. All of the.listed drugs are
frequently used
in combination with other drugs.
Other Pyrimidine Analogs
There are a large number of pyrimidine analogs in clinical development for a
wide variety of indications. One of the most common indications is cancer and
1o leukemia and lymphoma of various types. For example, 2',2'-
difluorodeoxycytidine
(gemcitabine; Gemzar) is a pyrimidine nucleoside drug with clinical efficacy
in
several common solid cancers; cytosine arabinoside (AItA-C) is another
pyrimidine
analog used in the treatment of leukemia; 2-chlorodeoxyadenosine and
fludarabine
(F-araA) are also used as antineoplastic drugs. 2'-deoxy-2'-(fluoromethylene)
15 cytidine (MDL 101,731, Kyowa Hakko Kogyo Co.), 2',2'-difluorodeoxycytidine,
5
aza-2'deoxycytidine (decitabine), 5-azacytidine, 5-azadeoxycytidine, and - are
under development as antineoplastic drugs.
CNS Drugs - Pyrimidine Pathway
2o The pyrimidine nucleoside, uridine, has been proposed as a potential
supplement in the treatment of psychosis based on its ability to reduce
haloperidol-
induced dopamine release. Thus, coadministration of uridine with haloperidol
might
enhance the antipsychotic action of standard neuroleptics, allowing for a
reduction in
dose and thereby a reduction in the frequency of side effects. The presumed
25 mechanism is interaction with dopamine or GABA neurotransmission. The
levels or
function of pyrimidine transporters or pyrimidine de novo or salvage
biosynthetic
enzymes, or pyrimidine catabolic enzymes may affect the action of
neuroleptics, or
their modulation by pyrimidine nucleosides or pyrimidine analogs.
3o Other Therapeutics Relevant to the Pvrimidine Pathway
Another possible mode of pyrimidine nucleotide action is via stimulation of
thromboxane A2 release from cultured glial cells. Uridine triphosphate,
uridine
diphosphate, cytidine triphosphate, and deoxythymidine triphosphate all induce
concentration-dependent increases in the release of thromboxane A2 from
cultured
35 giial cells, indicating a possible role in brain response to damage in
vivo.
CA 02335649 2001-O1-19
117
WO 00/04194 PCT/US99/16440
Other cancers such as head and neck, breast, pancreas, other gastrointestinal
cancers including stomach and intestinal may be directly targeted by
therapeutic
intervention that affects DNA methylation levels, pyrimidine synthesis,
transport,
and degradation pathways.
Many neurological diseases in both the CNS and the periphery may also be
affected by therapeutic intervention of DNA methylation, pyrimidine synthesis,
transport, and degradation pathways. Such intervention may be of therapeutic
benefit to halt, retard, and or reduce symptoms of these often debilitating
diseases.
Example 9 - Drugs That Affect the Folate and Pyrimidine Pathways
There are many potential candidate therapeutic interventions or drugs that
can affect the folate and pyrimidine pathways. Categories of these are 5-FU
prodrugs, drugs that affect DNA methylation pathways, and other drugs that
have
been developed for similar indications as 5-FU.
S-FU prodrugs
The clinical development and use of 5-FU prodrugs is further subject to
improvement by the methods of this invention. These drugs are generally
modified
fluoropyrimidines that require one or more enzymatic activation steps for
conversion
2o into 5-FU. The activation steps may result in prolonged drug half life
and/or
selective drug activation (i.e. conversion to S-FU) in tumor cells.
Examples of such drugs include capecitabine (Xeloda, Roche), a drug that is
converted to 5-FU by a three-step pathway involving Carboxylesterase 1,
Cytidine
Deaminase and Thymidine Phosphorylase. Another 5-FU prodrug is 5'deoxy 5-FU
(Furtulon, Roche) which is converted to 5-FU by Thymidine Phosphorylase and/or
Uridine Phosphorylase. Another 5-FU prodrug is 1-(tetrahydro-2-furanyl)-5-
fluorouracil (FT, ftorafur, Tegafur, Taiho - Bristol Myers Squibb), a prodrug
that is
converted to 5-FU by cytochrome P450 enzyme, CYP3A4.
3o Drugs acting on DNA methyation pathways
Antivirals
Herpes virus thymidine kinase phosphorylates many 5-substituted 2'-
deoxyuridines, analogs of thymidine (e.g., idoxuridine, trifluridine,
edoxudine,
brivudine) and 5-substituted arabinofuranosyluracil derivatives (e.g., 5-Et-
Ara-U,
3s BV-Ara-U, Cl-Ara-U). The 5'-monophosphates are further phosphorylated by
cellular enzymes to the 5'-triphosphates, which are usually competitive
inhibitors of
the viral-coded DNA polymerases.
CA 02335649 2001-O1-19
11$
WO 00/04194 PCT/US99/16440
Unlike herpes viruses, retroviruses including but not limited to human
immunodeficiency viruses do not encode specific enzymes required for the
metabolism of the purine or pyrimidine nucleotides to their corresponding 5'-
triphosphates. Therefore, 2',3'-dideoxynucleosides and acyclic nucleoside
phosphonates must be phosphorylated and metabolized by host cell kinases and
other enzymes of purine and/or pyrimidine metabolism. In this way, affecting
the
pyrimidine synthetic, transport, or degradation pathways by candidate
therapeutic
intervention may be therapeutic beneficial in treating retroviral infections.
Excamples of candidate antivirals that may be affected by alteration of
pyrimidine
synthetic, transport, or degradation pathwyas are azidothymidine (AZT),
acyclovir,
and ganciclovir. These and other drugs have been used both as antivirals and
antineoplastic agents.
Other Drugs Developed for Similar Indications as S-FU
A variety of drugs are being developed for similar indications as 5-FU,
and/or are being tested in combinations with 5-FU/leukovorin. These include
the
new platinum compound oxaliplatin (L-OHP) and the topoisomerase I inhibitors
irinotecan (CPT11, Pharmacia-UpJohn) and topotecan. The effective clinical
development or clinical use of these drugs may be enhanced by the methods of
this
2o invention. In particular, identification of patients likely to respond to 5-
FU with or
withour leukovorin, may be useful in selecting optimal responders to other
drugs.
Alternatively identification of patients likely to suffer toxic response to 5-
FU
containing regimens may allow identification of patients best treated with
other
drugs. Other drugs with activity against cancers usually treated with regimens
containing 5-FU (e.g. metastatic colon cancer) include Suramin, a bis-
hexasulfonated napthylurea; 6-hydroxymethylacylfulvene (HMAF; MGI 114);
LY295501; bizelesin (U-7779; NSC615291), ONYX-015; monoclonal antibodies
(e.g. 17-lA and MN-14), protein synthesis inhibitors such as RA 700, and
angiogenesis inhibitors such as PF 4. Still other drugs may prevent colorectal
cancer
3o by preventing the formation of colorectal polyps (eg, cyclooxygenase
inhibitors may
induce apoptosis of polyps).
Example 10
Protocol for Clinical Trial for Determining the Relationship Between Toxicity
of a Drug and Genetic Variances in Genes Related to the Action of the Drug
THIS EXAMPLE PROVIDES AN EXEMPLARY CLINICAL TRIAL AS A
CASE CONTROL STUDY WHICH INCLUDES EVALUATING THE EFFECTS
CA 02335649 2001-O1-19
119
WO 00/04194 PCT/US99/16440
OF SEQUENCE VARIANCES IN ENZYMES WHICH CAN MEDIATE THE
EFFECTS OF A KNOWN DRUG, IN THIS CASE IN AN ANTICANCER
TREATMENT. THE INFORMATION IN THE BACKGROUND SECTION OF
THIS PROTOCOL IS ALSO PROVIDED IN LARGE PART IN THE DETATILED
DESCRIPTION, BUT IS REPEATED HERE FOR COMPLETENESS OF THE
PROTOCOL DESCRIPTION.
PROTOCOL TITLE: Case-control study to determine the
relationship between toxicity of 5-fluorouracil (5-FU) given with folinic acid
1o (FA) to patients with solid tumors and DNA sequence variances in enzymes
that mediate the action of 5-FU and FA.
CA 02335649 2001-O1-19
120
WO 00/04194 PCT/US99/16440
II. SIGNATURE PAGE
to
Name, position, and address of individual approving protocol from study
sponsor.
is
Name, position, and address of individual approving protocol from study
sponsor.
CA 02335649 2001-O1-19
121
WO 00/04194 PCT/US99/16440
lll. TABLE OF CONTENTS
SIGNATURE PAGE 120
s TABLE OF CONTENTS 121
ACRONYMS AND ABBREVIATIONS 123
STUDY FLOW CHART 124
1. SUMMARY 125
2. INTRODUCTION 127
l0 2.1 Background 127
2.1.1 Potential for Improved Effectiveness of 5-FU and 5-FU/FA 127
2.1.2 Metabolic Pathways that Affect 5-FU/FA Action 130
2.1.3 Genetically Determined Variation in Response to 5-FU: Studies of
Dihydropyrimidine Dehydrogenase Deficiency 133
15 2.1.4 Variances in Genes That May Affect 5-FU/FA Action 134
2.1.5 Analysis of Haplotypes Increases Power of Genetic Analysis I 34
2.1.6 Biochemical Studies of Alternate Allelic Fotms of DPD 136
2.2 Study Rationale 137
3. OBJECTIVES 137
20 3.1 Primary Objective 137
3.2 Secondary Objectives 138
4. STUDY DESIGN 138
4.1 Study Outline 138
4.2 Subject Withdrawal from the Study 138
2s 4.3 Discontinuation of the Study 138
5. STUDY POPULATION 139
S.1 Number of Subjects 139
5.2 Inclusion Criteria 139
5.3 Exclusion Criteria 139
30 5.4 Screening Log 140
6. ALLOCATION PROCEDURE 140
8. SCHEDULE OF EVENTS 140
11. STATISTICAL STATEMENT AND ANALYTICAL PLAN 141
11.1 Sample Size Considerations 141
35 I 1.2 Description of Objectives and Endpoints 142
11.2.1 Primary Objective and Endpoints 142
11.2.2 Secondary Objectives and Endpoints 142
11.3 CRiteria for the Endpoints 143
CA 02335649 2001-O1-19
122
WO 00/04194 PCT/US99/1~440
11.4 Statistical Methods To Be Used in Objective Analyses 143
12. ETHICAL REQUIREMENTS 144
12.1 Declaration of Heisinki 144
12.2 Subject Information and Consent 144
12.3 Subject Data Protection 144
13. FURTHER REQUIREMENTS AND GENERAL INFORMATION
145
13.1 Study Committee 145
13.2 Changes to Final Study Protocol 145
l0 13.3 Record Retention 145
13.4 Reporting and Communication of Results 146
13.5 PROTOCOL COMPLETION 146
REFERENCES 147
SIGNED AGREEMENT OF THE STUDY PROTOCOL 149
is APPENDIX II 150
CA 02335649 2001-O1-19
123
WO 00/04194 PCT/US99/16440
IV. ACRONYMS AND ABBREVIATIONS
5-FU s-Fluorouracil
FA Folinic acid
s C Degree centigrade
CBC Complete blood count
CRF Case report form
DCC Data Coordinating Center
DMC Data Monitoring Conunittee
1 o EC Ethical Committee
ECG Electrocardiogram
e.g. For example
F Degrees Fahrenheit
FDA Food and Drug Administration
is i.e. That is
IRB Institutional Review
Board
IV Intravenous
mcg Microgram
mg Milligram
2o mL Milliliter
mm' Cubic millimeter
PD Pharmacodynamic
PK Pharmacokinetic
~ Registered trade mark
2s REB Research Ethics Board
USA United States of America
USP United States Pharmacopoeia
CA 02335649 2001-O1-19
124
WO 00/04194 P(_'T/IIC99It64.40
i! STUDYFLOW CHART
File Medical
Research Visit
Selection of atients from X
the file
Informed Consent Form si X
ed
Inclusion/Exclusion criteria X
checkin
Chart r ortin X
Demo a hic r ortin X
Blood sam lin X
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
125 . -
VI. !. SUMMARY
Protocol
Title: Case-control study to determine the relationship
between toxicity of 5-fluorouracil (S-FU) given with folinic acid (FA) to
patients with solid tumors and DNA sequence variances in enzymes that
mediate the action of 5-FU and FA.
VII. Stu~,Y
VIII. Phase: Phase IV
Study
Design: Single-center, case-control study.
Study
Objectives: The primary objective of this study is to compare the variance
frequency distribution in the dihydropyrimidine dehydrogenase (DPD) gene
between
two groups of patients with solid tumors, treated by weekly or monthly regimen
of
5-FU+FA and defined by level of toxicity (graded according to the NCI common
toxicity criteria) as:
- Group 1: patients with high toxicity (grade III / IV on NCI criteria)
- Group 2: patients with minimal toxicity (grade 0 / I / II on NCI criteria)
The secondary obJectives of the study are to determine the DPD gene haplotype
frequency distribution and the variance and/or haplotype frequency
distributions in
selected genes (other than DPD gene) between two groups of patients with solid
tumors, treated by weekly or monthly regimen of S-FU+FA and defined by level
of
toxicity. Analyses will be done globally, then by regimen (monthly vs. weekly)
and by
type of toxicity (gastrointestinal vs. bone marrow).
Number of Subjects: Ninety (90) patients, 45 in each group, will be included.
Study Population: Patients treated with 5-FU+FA for solid tumors at the
Massachusetts General Hospital, Dana-Farber Cancer Institute and Brigham and
Women's Hospital.
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
126 . -
StudyGroups: Patients will be divided into two groups depending on the degree
of
toxicity they experienced with treatment, if any:
- patients with high toxicity (grade III / IV on NCI criteria),
- patients with minimal toxicity (grade 0 / I / II on NCI criteria)
Visit Schedule: One visit to sign the informed consent form and to collect
blood sample.
Evaluation Parameter: Frequency distribution of gene alleles and haplotypes.
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
127 . w
IX. Z. INTRODUCTION
X. 2.1 Background
XI. 2.1.1 Potential for Improved Effectiveness of 5-FU and 5-FU/FA
Introduction
l0 Chemotherapy of cancer involves use of highly toxic drugs with nan'ow
therapeutic
indices. Although progress has been made in the chemotherapeutic treatment of
selected malignancies, most adult solid cancers remain highly refractory to
treatment. Nonetheless, chemotherapy is the standard of care for most
disseminated
solid cancers. Chemotherapy often results in a significant fraction of treated
patients
15 suffering unpleasant or life-threatening side effects while receiving
little or no
clinical benefit; other patients may suffer few side effects and/or have
complete
remission or even cure. Any test that could predict response to chemotherapy,
even
partially, would allow more selective use of toxic drugs, and could thereby
significantly improve efficacy of oncologic drug use, with the potential to
both
2o reduce side effects and increase the fraction of responders. Chemotherapy
is also
expensive, not just because the drugs are often costly, but also because
administering
highly toxic drugs requires close monitoring by carefully trained personnel,
and
because hospitalization is often required for treatment of (or monitoring for)
toxic
drug reactions. Infonmation that would allow patients to be divided into
likely
25 responder vs. non-responder (or likely side effect) groups, only the former
to receive
treatment, would therefore also have a significant impact on the economics of
cancer
drug use.
Predicting Response to Chemotherapy
Several methods for predicting response to chemotherapy in individual patients
have
been investigated over the years, ranging from the use of biochemical markers
to
testing drugs on a patients cultured tumor cells. None of these methods has
proven
sufficiently informative and practical to gain wide acceptance. However, there
are
some specific examples of tests useful for predicting toxicity. For example, a
diagnostic test to predict side effects associated with the antineoplastic
drugs 6-
mercaptopurine, 6-thioguanine and azathioprine has begun to gain wide
acceptance,
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
128
particularly among pediatric oncologists. Severe toxicity of thiopurine drugs
is
associated with deficiency of the enzyme thiopurine methyltransferase (TPMT).
Currently most TPMT testing is done using an enzyme assay, however the TPMT
gene has been cloned and mutations associated with low TPMT levels have been
identified; genetic testing is beginning to supplant enzyme assays because
genetic
tests are more easily standardized and economical.
While there are no good tests that predict positive chemotherapeutic response,
there
is demonstrated utility to measuring estrogen and progesterone receptor levels
in
to cancer tissue before selecting therapy directed at modulating honmonal
state.
Measuring genetic variation in proteins that mediate the effects of
chemotherapy
drugs is in some respects analogous to measuring ER and PR levels, which
mediate
the effects of hormones.
15 Clinical Use and Efj''ectiveness of S-FU and S-FUlFA
5-FU is a pyrimidine analog in clinical use since 1957. 5-FU is used in the
standard
treatment of gastrointestinal, breast and head and neck cancers. Clinical
trials have
also shown responses in cancer of the bladder, ovary, cervix, prostate and
pancreas.
2o The remainder of this discussion will concern colorectal cancer. 5-FU is
used both
in the adjuvant therapy of Dukes Stage B and C cancer and in the treatment of
disseminated cancer. 5-FU alone produces partial remissions in 10 - 30% of
advanced colorectal cancers, however only a few percent of patients have
complete
remissions. In the last 1 S years a variety of biochemically motivated
strategies for
25 modulating 5-FU activity have been tested. For example, 5-FU has been used
in
combination with PALA, a pyrimidine synthesis inhibitor, to deplete cellular
pools
of UTP and thereby enhance formation of FUTP; in combination with
methotrexate,
to inhibit purine anabolism, leading to increased PRPP levels and consequent
increased conversion of 5-FU to its active nucleotide metabolites; and in
3o combination with folinic acid, which increases intracellular pools of
reduced folate,
driving formation of the ternary inhibitory complex formed by 5,10
methylenetetrahydrofolate, FdUMP and thymidylate synthase. Levamisole,
interferon and alkylating agents have also been used in combination with 5-FU.
5-
FU/Levamisole and 5-FU/FA are widely used in the adjuvant treatment of colon
35 cancer, while 5-FU/FA is the most commonly used regimen for advanced
colorectal
cancer. Several prospective randomized trials of 5-FU/FA vs. 5-FU alone in
patients
with advanced cancer have demonstrated up to two fold higher response rates to
5-
CA 02335649 2001-O1-19
WO 00/04194 PCTNS99/16440.
129
FU/FA, while three of the studies also showed increased survival. Two major
dosing regimens are used: 5-FU plus low dose FA given for five consecutive
days
followed by a 23 day interval, or once weekly bolus IV 5-FU plus high dose FA.
The higher FA dose results in plasma FA concentrations of 1 to 10 uM,
comparable
to those required for optimal 5-FU/FA synergy in tissue culture, however low
dose
FA (20 mg/mz vs. 500 mg/m~) has produced comparable clinical benefit. Ongoing
clinical trials are designed to further test new drug combinations. In
summary,
relatively few patients - in the single digits - live longer as a result of 5-
FU/FA,
although significantly more have partial disease rerziission. The factors that
to determine which patients respond or have side effects are not known.
Toxicity of S-FU and Folinic Acid
5-FU toxicity has been well documented in randomized clinical trials. Patients
receiving 5-FU/FA are at even greater risk of toxic reactions and must be
monitored
carefully during therapy. A variety of side effects have been observed,
affecting the
gastrointestinal tract, bone marrow, heart and CNS. The most common toxic
reactions are nausea and anorexia, which can be followed by life threatening
mucositis, enteritis and diarrhea. Leukopenia is also a problem in some
patients,
2o particularly with the weekly dosage regimen. In a recent randomized trial
of weekly
vs. monthly 5-FU/FA there were 7 deaths related to drug toxicity among 372
treated
patients ( 1.9%; Buroker et al. 1994). 3 t % of patients receiving the weekly
regimen
suffered diarrhea-requiring hospitalization for a median of 10 days. Other
severe
toxicity, which occurred at lower frequency, included leukopenia and
stomatitis. In
another example, 36% of patients receiving weekly bolus 5-FU pius FA (500
mg/m2), in a NSABP trial suffered NCI grade 3 toxicity (Wolmark et al., 1996).
Clearly, toxicity is a major cost of 5-FU/FA therapy, measured both in patient
suffering and in financial terms (the cost of care for drug induced illness).
Other Factors
Many non-genetic factors influence the response of cancers to drugs, including
tumor location, vasculature, cell growth fraction and various drug resistance
mechanisms. It will therefore not be possible to explain all heterogeneity in
response to 5-FU/FA by genetic variation. However, based on genetic studies of
other quantitative traits it seems likely that a significant fraction of
variation in drug
response can be explained (see below).
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
130
XII. 2.1.2 Metabolic Pathways that Affect 5-FUIFA Action
The biochemical pathways of 5-FU metabolism have been studied extensively.
Likewise, folate metabolism has been well investigated and the enzymes that
form
and consume 5, 10-methylenetetrahydrofolate are well known. The principal
metabolic pathways that influence the pharmacologic action of 5-FU are
summarized in Figure 1.
Figure 1. 5-FU metabolism and inhibition of thymidylate formation. Enzymes:
1. uridine phosphorylase; 2. thymidine phosphorylase; 3. orotate
phosphoribosyl
transferase; 4. thymidine kinase; S. uridine kinase; 6. ribonucleotide
reductase; 7.
thymidylate synthase; 8. dCMP deaminase; 9. nucleoside monophosphate kinase;
10. nucleoside diphosphate kinase; 11. nucleoside diphosphatase or cytidylate
is kinase; 12: thymine phosphorylase. FH2 = dihydrofolate, FH4 =
tetrahydrofolate.
The Figure is adapted from Goodman & Gilinan's The Pharmacological Basis of
Therapeutics, ninth edition, McGraw Hill, 1996, p. 1249.
De novo and salvage routes of pyrimidine nucleotide formation (S-FU anabolism)
2o and inhibition of thymidylate synthase
5-FU is a biologically inactive pyrimidine analog, which must be
phosphorylated,
and ribosylated to the nucleoside analog fluorodeoxyuridine monophosphate
(FdUMP) to have clinical activity. FdUMP formation can occur via several
routes,
25 summarized in Figure 1. 5-FU may be converted by uridine phosphorylase to
fluorouridine (FLJdR; the reverse reaction is catalyzed by uridine
nucleosidase) and
then to fluorouridine monophosphate (FLJMP) by uridine kinase, or FUMP may be
formed from 5-FU in one step via transfer of a phosphoribosyl group from 5-
phosphoribosyl-1-pyrophosphate (PRPP), catalyzed by orotate phosphoribosyl
30 transferase. FLTMP can be converted to FUDP and subsequently FUTP by a
nucleoside monophosphate kinase and nucleoside diphosphate kinase,
respectively.
FUTP is incorporated into RNA by RNA polymerises, which may account in part
for 5-FU toxicity as a result of effects on processing or function (e.g.
translation).
Alternatively, FUDP may be reduced to the dinucleotide level, FdUDP
35 (fluorodeoxyuridine diphosphate) by ribonucleotide diphosphate reductase, a
heterodimeric enzyme. FdUDP can then be converted to FdUTP by nucleoside
diphosphate kinase and incorporated into DNA by DNA polymerises, which may
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
131
account for some 5-FU toxicity. Fluoropyrimidine modified.DNA may also be
targeted by the nucleotide excision repair process. The more important path of
FdUDP metabolism with respect to anticancer effects, however, is believed to
be
conversion to FdUMP by nucleoside diphosphatase (or cytidylate kinase, a bi-
directional enzyme). dUMP is the precursor of dTMP in de novo pyrimidine
biosynthesis, a reaction catalyzed by thymidylate synthase and which consumes
5,10-methylenetetrahydrofolate, producing 7,8 dihydrofolate. FdUMP, however,
forms an inhibitory (probably covalent) complex with thymidylate synthase in
the
presence of 5,10-methylenetetrahydrofolate, therebyblocking formation of
to thymidylate (other than by the salvage pathway via thymidine kinase). The
complex
anabolism of FdUMP can be simplified by giving the deoxyribonucleoside of 5-
FU,
5-fluorodeoxyuridine (also called floxuridine; FUdR), which can be converted
to
FdUMP in one step by thymidine kinase. However, FUdR is also rapidly converted
back to 5-FU by the bi-directional enzyme thymidine phosphorylase.
S-FU catabolism.
Metabolic elimination of 5-FU occurs via a three-step pathway leading to -
alanine.
The first and rate limiting enzyme in the elimination pathway is
dihydropyrimidine
dehydrogenase (DPD), which transforms more than 80% of a dose of 5-FU to the
inactive dihydrofluorouracil form. Subsequently dihydropyrimidinase catalyzes
opening of the pyrimidine ring to form 5-fluoro--ureidopropionate and then -
ureidopropionase (also called -alanine synthase) catalyzes formation of 2-
fluoro--
alanine. The first two reactions are reversible. The distribution of activity
of these
enzymes in human populations has not been established, however, a recent
population survey of urinary pyrimidine levels in 1,133 adults revealed that
levels of
dihydrauracil range from 0 - 59 uM/g of creatinine, while uracil levels ranged
from 0
- 130 uM/g creatinine (Hayashi et al., 1996), suggesting variation in the
activity of
enzymes of pyrimidine metabolism. It is worth noting that in animal studies
3o catabolites of 5-FU apparently account for some fraction of 5-FU toxicity
(Davis et
al., 1994; Spector et al., 1995). This result is the rationale for current
human trials of
5-FU combined with DPD inhibitors: if the 5-fluoro- metabolites are
responsible for
toxicity, then blocking their formation by inhibition of DPD, while
simultaneously
decreasing 5-FU dosage to compensate for the block in catabolism and
excretion,
should result in a better therapeutic index.
Folinic acid conversion to tetrahvdrofolate.
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440.
132
The conversion of FA to 5, l OMTHF can occur via several routes, illustrated
in
Figure 2
Figure 2. Folate metabolism and formation of 5,10-metbylenetetraiiydrofotate.
Enzymes: 1. Formimino-tetrahydrofolate cyclodeaminase; 2.
methenyltetrahydrofolate synthetase; 3. methenyltetrahydrofolate
cyclohydrolase; 4.
formyltetrahydrofolate synthetase; 5. formyltetrahydrofolate hydrolase; 6.
fortnyltetrahydrofolate dehydrogenase; 7. methylenetetrahydrofolate
dehydrogenase;
to 8. methylenetetrahydrofolate reductase (MTHFR); 9. homocysteine
methyltransferase (also called methionine synthetase); 10. serine
transhydroxymethylase; 11. glycine cleavage system; 12. thymidylate synthase;
13.
dihydrofolate reductase. Abbreviations: THF = tetrahydrofolate; DHF =
dihydrofolate. Note that THF appears twice (i.e. the product of step 6 is also
substrate for enzymes 10 and 11. Step 12 also appears in Figure 1, above. This
Figure is adapted from Mathews & van Holde, Biochemistry, The
Benjamin/Cummings Publishing Co., Redwood City CA, 1990, page 697.
Intracellular reduced folate levels can potentiate 5-FU action by increasing
5,10-
methyl-enetetrahydrofolate levels (5,10-methyleneTHF; see center of Figure 2),
thereby stabilizing the ternary inhibitory complex formed with thymidylate
synthase
and FdUMP. This is the basis for therapeutic modulation of 5-FU with FA. As
can
be seen in Figure 2, conversion of foIinic acid (5-formylTHF) to 5,10-
methenylTHF,
the precursor of 5,10-methyleneTHF, requires methenyltetrahydrofolate
synthetase
(enzyme 2 in the Figure). Also, levels of 5,10-methyleneTHF may be affected
directly by the activity of methylenetetrahydrofolate dehydrogenase,
methylenetetrahydrofolate reductase, serine transhydroxymethylase and the
glycine
cleavage system enzymes (7, 8, 10 and 11 in Fig. 2), and indirectly by the
other
enzymes shown in the Figure.
Cell uptake ofpyrimidine nucleosides and folinic acid
Human cells have five concentrative nucleoside transporters with varying
patterns of
tissue distribution (see review by Wang et al., 1997). Two transporters, one
with
preference for purines and one for pyrimidines have been cloned recently
(Felipe et
al., 1998). 5-FU entry into cells may be modulated by activity of these
transporters,
particularly the pyrimidine transporter, although one prospective randomized
clinical
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
133
trial in which the nucleoside transport inhibitor dipyridamole was paired with
S-FU
and FA failed to show a difference in outcome compared to 5-FU/FA alone (Kohne
et al., 1995). Several folate transport systems have been identified in human
cells.
Folate receptor 1 (FR1 ) is a high affinity (nanomolar range) receptor for
reduced
folates. Three restriction fragment length polymorphisms (RFLPs) have been
reported at the FR1 locus (Campbell et al., 1991). Reduced folates are also
transported by folate receptor gamma and by a low affinity ( 1 uM) folate
transporter.
15-fold variations in levels of folate transporter have been described in
unselected
tumor cell lines (Moscow et al., 1997).
to
XIII. 2.1.3 Genetically Determined Variation in Response to 5-FiJ: Studies of
Dihydropyrimidine De6ydrogenase Deficiency
Dihydropyrimidine Dehydrogenase Deficiency is Associated with S-FU Toxicity
!5
5-FU is inactivated by the same metabolic pathway as thymine and uracil (see
above). DPD catalyzes the first, rate-limiting step in pyrimidine catabolism
and
accounts for elimination of most 5-FU. Normal individuals eliminate 5-FU with
a
half life of ~10-15 minutes and excrete only 10% of a dose unchanged in the
urine.
2o In contrast, people genetically deficient in DPD eliminate 5-FU with a half
life of
~2.5 hours and excrete 90% of a dose unchanged in the urine (Diasio et al., I
988).
DPD deficiency has two clinical presentations: (i) an inborn error of
metabolism
causing some degree of neurologic dysfimction or (ii) asymptomatic until
revealed
by exposure to 5-FU or other pyrimidine analogs. With either presentation
there is
25 combined hyperuracilutia and hyperthyminuria. The vastly increased 5-FU
half life
in DPD deficient individuals causes severe toxicity and even death. Recently
several
mutations have been identified in DPD genes of deficient individuals (Wei et
al.,
1996), however none of these alleles appears to occur at appreciable
frequency, so
the cause of wide population variation in DPD levels is still not understood.
Population Studies of DPD Activity Show Wide Variation
Population surveys of DPD activity in normal individuals have been performed
using blood and liver samples. These studies reveal a broad unimodal Gaussian
distribution of DPD activity over a 7 to 14 fold range, with some individuals
having
very low or even undetectable levels. For example Etienne et al. ( 1994)
report DPD
activity ranging from .065 to .559 nM/min/mg protein in a study of 152 men and
33
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
134
women, while Fleming et al. ( 1993) found DPD activity in 66 cancer patients
varied
from .17 to .77 nM/min/mg protein. Lu et al (1995) found 18-fold variation in
liver.
DPD assayed in 138 individuals. Milano and Etienne (1994) suggested that the
frequency of heterozygous and homozygous deficiency is 3% and .I %,
respectively.
The DNA sequence alterations responsible for null DPD aileles do not account
for
the high population variability (Ridge et al., 1997).
DPD Levels are correlated with Response to 5-FU
to Intratumoral DPD levels have been measured in patients receiving S-FU
chemotherapy. When complete responders were compared to partial or non-
responders, DPD levels were lower in the compete responders (Etienne et al.,
1995).
Leukocyte DPD levels has also been measured in patients receiving 5-FU/FA
chemotherapy. When patients were divided into 3 groups: high, medium and low
15 DPD activity, the frequency of serious side effects was highest in the low
DPD
group and vice versa (Katona et al., 1997).
XIV. 2.1.4 Variances in Genes That May Affect 5-FU/FA Action
2o Variagenics has already surveyed thymidylate synthase, ribonucleotide
reductase
(M1 subunit only), and dihydrofoiate reductase and dihydropyrimidine
dehydrogenase cDNAs for genetic variation. 36 unrelated individuals were
screened
using 6 SSCP conditions and DNA sequencing. Other investigators have
identified
variances in MTHFR, methionine synthase and folate receptor. These findings
are
25 summarized in Appendix I.
XV.
XVI. 2.1.5 Analysis of Haplotypes Increases Power of Genetic Analysis
It is evident from work to date that, while DPD activity is weakly predictive
of 5-FU
3o toxicity and drug response, there must be other factors that account for
some of the
variation in patient response. This is to be expected as drug response
phenotypes
usually vary continuously, and such (quantitative) traits are typically
influenced by a
number of genes (Falconer and Mackay, 1997). Although it is impossible to
determine a priori the number of genes influencing a quantitative trait, often
only a
35 few loci have large effects, where a large effect is 5-20% of total
variation in the
phenotype (Mackay, 1995).
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
135
Having identified genetic variation in enzymes that may affect 5-FU action,
how can
we most efficiently address its relation to phenotypic variation? The
sequential
testing for correlation between phenotypes of interest and single nucleotide
polymorphisms may be adequate to detect associations if there are major
effects
associated with single nucleotide changes; certainly it is worth performing
this type
of analysis. However there is no way to know in advance whether there are
major
phenotypic effects associated with single nucleotide changes and, even if
there are,
there is no way to be sure that the salient variance has been identified by
screening
cDNAs. A more powerful way to address the question of genotype-phenotype
to correlation is to assort genotypes into haplotypes. (A haplotype is the cis
arrangement of polymorphic nucleotides on a particular chromosome.) Haplotype
analysis has several advantages compared to the serial analysis of individual
polymorphisms at a locus with multiple polymorphic sites.
(1) Of all the possible haplotypes at a locus (2° haplotypes are
theoretically
possible at a locus with n binary polymorphic sites) only a small fraction
will
generally occur at a significant frequency in human populations. Thus,
association
studies of haplotypes and phenotypes will involve testing fewer hypotheses. As
a
result there is a smaller probability of Type I errors, that is, false
inferences that a
particular variant is associated with a given phenotype.
(2) The biological effect of each variance at a locus may be different both in
magnitude and direction. For example, a polymorphism in the 5' UTR may affect
translational efficiency, a coding sequence polymorphism may affect protein
activity, a polymorphism in the 3' UTR may affect mRNA folding and half life,
and
so on. Further, there may be interactions between variances: two neighboring
polymorphic amino acids in the same domain - say cys/arg at residue 29 and
met/val
at residue 166 - may, when combined in one sequence, for example, 29cys-
166va1,
have a deleterious effect, whereas 29cys-166met, 29arg-166met and 29arg-166va1
3o proteins may be nearly equal in activity. Haplotype analysis is the best
method for
assessing the interaction of variances at a locus.
(3) Templeton and colleagues have developed powerful methods for assorting
haplotypes and analyzing haplotype/phenotype associations (Templeton et al.,
1987). Alleles, which share common ancestry, are arranged into a tree
structure
(cladogram) according to their time of origin in a population. Haplotypes that
are
evolutionarily ancient will be at the center of the branching structure and
new ones
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
136 _
(reflecting recent mutations) wilt be represented at the periphery, with the
links
representing intermediate steps in evolution. The cladogram defines which
haplotype-phenotype association tests should be performed to most efficiently
exploit the available degrees of freedom, focusing attention on those
comparisons
most likely to define functionally different haplotypes (Haviland et al.,
1995). This
type of analysis has been used to define interactions between heart disease
and the
apolipoprotein gene cluster (Haviland et al 1995) and Alzheimer's Disease and
the
Apo-E locus (Templeton 1995) among other studies, using populations as small
as
50 to 100 individuals.
XVII. 2.1.6 Biochemical Studies of Alternate Allelic Forms of DPD
The power of genetic analysis can be augmented by biochemical studies of
alternate
allelic forms of enzymes. Biochemical data on the distribution of activity of
a series
of enzymes in a biochemical pathway provides the basis for metabolic flux
analysis
(Keightly, 1996). It is beyond the scope of this clinical trial to analyze
biochemical
variation in the enzymes of pyrimidine and folate metabolism. However, since
Variagenics has identified new variances in DPD that may plausibly affect
enzyme
expression or activity, and because DPD is already proven to play a role in 5-
FU
2o response, parallel studies will be conducted to investigate the
relationship between
genotype and biochemistry for this enzyme.
DPD cDNAs have been cloned from a variety of higher eukaryotes and binding
sites
for its cofactors, prosthetic groups and substrate have been defined
experimentally or
by analogy with known consensus motifs (Yokata et al., 1994). The DPD
polymorphisms that affect protein sequence occur at amino acids 29 (cys/arg)
and
166 (met/val) in the amino-terminal one-third of the protein. Phylogenetic
comparison of this region from boar, human, cow, fly, and bacteria (see below)
shows that there are actually two highly conserved motifs that resemble either
3o iron/sulfur or zinc binding motifs, the latter being more likely due to the
spacing of
the cysteine residues. The region around the met/val polymorphism at amino
acid
166 is highly conserved. Even the spacing of the putative zinc-finger domains
is
maintained between distantly related species, hinting at their importance.
Since
amino acid 166 is close to a highly conserved (and probably functionally
important)
region and is itself conserved, being a methionine in all species, it seems
likely that
perturbations in this position would have consequence. The polymorphism
substitutes a long amino acid side chain capable of hydrogen bonding
(methionine)
CA 02335649 2001-O1-19
WO 00/04194 PCTIUS99/16440
137
for a compact, hydrophobic amino acid (valine). The region around amino acid
29 is
not as well conserved.
~VIIL 2.2 Study Rationale
5-fluorouracil (5-FU) is a fluorinated pyrimidine analog that is widely used
in
chemotherapy. The effectiveness of 5-FU is potentiated by folinic acid (FA:
generic
name: leukovorin). The combination of 5-FU and FA is standard therapy for
stage
III/IV colon cancer. Patient responses to 5-FU and 5-FU/FA vary widely,
ranging
1o from complete remission of cancer to severe toxicity.
- Pyrimidine base analogs are degraded by the same enzymes that degrade
endogenous uracil and thymine. Dihydropyrimidine dehydrogenase (DPD) is the
first degradative enzyme in this pathway, accounting for catabolism of more
than
t 5 80% of an administered dose of 5-FU.
Total DPD deficiency (familial pyrimidinemia and pyridinuria) is a rare
syndrome
associated with 5-FU induced toxicity. A milder defect in DPD activity appears
to
account for the severe side effects that occur in 1%-3% of unselected cancer
patients
20 (Milano and Etienne, 1994).
The major toxic manifestations of 5-FLT and FA depend on the schedule of
administration and occur mainly in rapidly dividing tissues such as bone
marrow and
the mucosal lining of the gastrointestinal tract.
This study is designed to test whether genetically encoded biochemical
variations in
the enzymes of pyrimidine catabolism, nucleotide metabolism and folic acid
metabolism, among patients treated with a weekly or monthly schedule of 5-
FU+FA,
account for some of the variation in drug toxicity. Applications of a
successful
3o pharmacogenetic study lie in the direction of safer, more efficacious, and
hence more
economical use of 5-FU, guided by genetic tests.
XIX. 3. OBJECTIVES
XX. 3.1 Primary Objective
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440.
138
The primary objective of this study is to compare the variance frequency
distribution in the dihydropyrimidine dehydrogenase (DPD) gene between two
groups of patients with solid tumors, treated by weekly or monthly regimen of
5-
FU+FA and defined by level of toxicity (graded according to the NCI common
toxicity criteria) as:
- Group 1: patients with high toxicity (grade III / IV on NCI criteria)
- Group 2: patients with minimal toxicity (grade 0 / I / II on NCI criteria)
XXI. 3.2 Secondary Objective8
IO
The secondary objectives of the study are to determine the DPD gene hapiotype
frequency distribution and the variance and/or haplotype frequency
distributions in
selected genes (other than DPD gene -see Appendix I-) between two groups of
patients
with solid tumors, treated by weekly or monthly regimen of 5-FU+FA and defined
by
level of toxicity. Analyses will be done globally, then by regimen (monthly
vs. weekly)
and by type of toxicity (gastrointestinal vs. bone marrow).
2o XXII. 4. STUDYDESIGN
XXIII.4.1 Study Outline
The study will be done at selected medical institution.
The study is a single-center, case-control study. The duration of the study is
expected to be not more than 8 months.
Genetic analysis of anonymized patient samples will take place at the study
sponsor.
~C.~!CIV. 4.2 Subject Withdrawal from the Studx
Subjects who desire to discontinue participation in this study must be
withdrawn
from the study.
XXV. 4.3 Discontinuation of the Studv
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
139
This study may be terminated by the study sponsor, after consultation with the
Advisory Committee (see Section I 1.2), at any time.
XXVl. S. STUDYPOPULATION
XXVII. 5.1 Number of Subjects
Ninety (90) subjects will be recruited for the study.
to
XXVIII. 5.2 Inclusion Criteria
To be eligible for entry into this study, candidates must meet the following
eligibility criteria at the time of enrollment:
is
1. Above age of 18 years.
2. Diagnosis of solid tumor.
2o 3. Treatment with a weekly or monthly regimen of 5-fluorouracil (5-FLI)
plus
folinic acid (FA)
4. Classified according to the NCI common toxicity criteria as 0, I, II, III
or IV
grade.
5. Give written infonmed consent prior to any testing under this protocol,
including
screening tests and evaluations that are not considered part of the subject's
routine
care.
XXIX. 5.3 Exclusion Criteria
Candidates will be excluded from study entry if any of the following exclusion
criteria exist at the time of enrollment:
Medical History
1. Diagnosis of cancer other than solid tumor.
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
140
2. Classified according to the NCI common toxicity criteria as grade iI.
3. Known history of HIV, HBV or Hepatitis C virus infection (undesirable for
making permanent cell line).
Treatment History
4. Treatment with 5-FU + FA but with other schedule than weekly or monthly.
S. Concomitant treatment with other cancer drugs than S-FU+FA.
Miscellaneous
6. Unwillingness or inability to comply with the requirements of this
protocol.
XXX. 5.4 Screening Log
For every patient initially considered for inclusion in this study, it is
required to
2o document and to specifically state the reasons) for their exclusion.
X.Y~ 6. ALLOCATIONPROCEDURE
When the eligibility review screening has been completed and the subject has
been
found eligible for admission to the study, the subject will be assigned to one
of the
two following group, depending on the 5-FU+FA related toxicity. he has
experienced
in the past:
- Group 1: patients with high toxicity (grade III / IV on NCI criteria)
- Group 2: patients with minimal toxicity (grade 0 / I / II on NCI criteria)
7. SCHEDULE OF EVENTS
XXXll. Parfenu
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
141
Patients will only be required to come for giving informed consent, then
having one
blood drawing ( 17ml total} -see Appendix II-.
Studv Personnel
The following personnel will be involved in the conduct of this study.
~ A treating physician who will oversee subject assignment and discuss the
protocol with the subject in order to obtain informed consent.
~ A treating nurse who will assist the treating physician in subject
identification
management and perform blood sampling.
~ A data manager who will collect and enter data in the clinical database.
Tests and Evaluations
The tests and evaluations described below must be performed by the reguired
study
personnel in order to determine subject eligibility.
Treating physician
~ Chart and demographic (sex, age, etc) reporting, inclusion/exclusion
criteria
checking.
Treating nurse
~ Blood sampling
Data manager
~ Clinical data entry.
XXXIII. Il. STATISTICAL STATEMENT AND ANALYTICAL
PLAN
XXJ!~IV. 11.1 Sample Size Considerations
The primary endpoint of this study is to measure and compare
genotype distributions of the DPD gene in patients with and without 5-
FU+FA toxicity. In order to be able to make a sample size calculation, we
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
142
will ignore the complexities of the underlying genetic model and treat the
data as n independent ordinary 2 x 2 contingency tables for the n variances in
the cases and controls. So, using the 2 most frequent DPD variances listed in
Appendix 1 and an odds-ratio of 4.00 for cases vs. controls, we can
determine the sample size for every variance, with an equal number of
subjects in each phenotypic (i.e. toxicity) group, required to detect, with
80%
power at a two-sided significance level of 0.05, a statistically significant
difference between distributions:
- nucleotide 3925; 44 patients per group
- nucleotide 3937: 43 patients per group.
A total of 90 patients (45 per group) will so be recruited.
11 2 Description of Objectives and Endpoints
XXXV. 11.2.1 Primary Objective and Endpoints
The primary objective of this study is to compare the variance frequency
distributions in the dihydropyrimidine dehydrogenase (DPD) gene between two
2o groups of patients with solid tumors, treated by weekly or monthly regimen
of 5-
FU+FA and defined by level of toxicity (grade 0/I/II vs. grade III/IV).
XXXVI. 11.2.2 Secondary Objectives and Endpoints
The secondary objectives of the study are:
1. To determine which DPD gene variances) is(are) associated to 5-FU+FA
toxicity
2. To determine which DPD haplotype(s) is(are) associated to S-FU+FA
toxicity.
3. To determine if one or more of the other gene variances (see Appendix 1)
is(are) associated to 5-FU+FA toxicity
4. To determine if one or more of the other haplotypes is(are) associated to 5-
FU+FA toxicity.
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
143
11.3 CRiteria for the End o
Since we do not know the mode of inheritance of a potential toxic
susceptibility, we
will ignore in a first step the complexities of the underlying genetic model
and treat
the data as an ordinary n x 2 contingency table for the n variances in the
cases and
controls.
Then, for every variance, we will compare genotype frequencies in order to
detect a
io potential effect of homo- vs. heterozygosity.
We will also compare haplotype frequencies of r predetermined haplotypes. The
method of cladograms (Templeton et al., 1987) will be used in an attempt to
find out
the smallest possible number r. In this method the evolutionary relationships
between present day haplotypes are represented as a tree or cladogram.
x;XXVII 11.4 Statistical Methods To Be Used in Objective Analyses
The statistical significance of the difference between variance frequencies
will be
assessed by a Pearson chi-squared test of homogeneity of proportions with n-1
degrees of freedom. Then, in order to determine which variances) is(are)
responsible for an eventual significance, we will consider each variance
individually
against the rest, yielding up to n comparisons each based on a 2 x 2 table.
This
should result in chi-squared tests that are individually valid but taking the
most
25 significant of these tests is a form of multiple testing. A Bonferroni's
adjustment for
multiple testing will so be made to the P-values such as p' = 1-(1-p)".
The statistical significance of the difference between genotype frequencies
associated to every variance will be assessed by a Pearson chi-squared test of
3o homogeneity of proportions with 2 degrees of freedom, using the same
Bonferroni's
adjustment as above.
Testing for unequal haplotype frequencies between cases and controls can be
considered in the same framework as testing for unequal variance frequencies
since a
35 single variance can be considered as a haplotype of a single locus. The
relevant
likelihood ratio test compares a model where two separate sets of haplotype
frequencies apply to the cases and controls, to one where the entire sample is
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
1~ _ _
characterized by a single common set of haplotype frequencies. This can be
performed by repeated use of a computer program (Terwilliger and Ott, 1994) to
successively obtain the log-likelihood corresponding to the set of haplotype
frequency estimates on the cases (ln L~,~), on the controls (ln L«"ro,) and on
the
overall (ln L«"b;"~). The test statistic 2(ln L~,~+ In L~""~, - In L~~,~,."~)
is then a chi-
squared with r -1 degrees of freedom (where r is the number of hapiotypes).
To test for potential confounding effects or effect-modifiers, such as sex,
age, etc.
logistic regression will be used with case-control status as the outcome
variable, and
genotypes and covariates (plus possible interactions) as predictor variables.
XXXYIII. 12. ETHICAL REQUIREMENTS
XXXIX. 12.1 Declaration of Helsinki
See Appendix III.
XL. 12.2 Subject Information and Consent
Prior to any testing under this protocol, including screening tests and
evaluations,
written informed consent must be obtained from the subject in accordance with
the
Standards of the Partners Cancercare Human Protection Committee (HPC).
The background of the proposed study and the benefits and risks of the
procedures
and study will be explained to the subject. A copy of the informed consent
document signed and dated by the subject must be given to the subject
Confirmation of a subject's informed consent must also be documented in the
subject's medical records prior to any testing under this protocol, including
3o screening tests and evaluations.
XLI. 12.3 Subject Data Protgction
The subject will not be identified by name or other any identifying
characteristic in
any study reports, and these reports will be used for research purposes
only.the study
sponsor, its designee(s), and various Government Health Agencies may inspect
the
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
145
records of this study. All relevant demographic and historical data regarding
patient
drug response will be recorded in an anonymized database.
XLII. 13. FURTHER REQUIREMENTS AND GENERAL
INFORMATION
XLIIL 13.1 Study Committee
l0 Advisory Committee
An Advisory Committee will be formed to provide scientific and medical
direction
for the study and to oversee the administrative progress of the study. The
Advisory
Committee will meet at least once a month to monitor subjects. The Advisory
Committee will determine whether the study should be stopped or amended for
any
reason.
The Advisory Committee will be comprised of the Director of Clinical
Pharmacogenetics, Vice-President for Discovery Research from the study sponsor
(and/or their designee) and participating investigators. The principal
investigator will
chair the Advisory Committee.
IiI.,IV. 13.2 rhanEes to Final Study Protocol
All protocol amendments must be submitted to the IRB/REB/EC. Protocol
modifications that impact on subject safety, the scope of the investigation,
or affect
the scientific quality of the study must be approved by the IRB/REB/EC and
submitted to the appropriate regulatory authorities before initiation.
However,
Variagenics may, at any time, amend this protocol to eliminate an apparent
immediate hazard to a subject. In this case, the appropriate regulatory
authorities
3o will be subsequently notified. In the event of a protocol modification, the
subject
consent form may require similar modifications.
XLV. I3.3 Record Retention
The Principal Investigator must maintain the records of signed consent forms,
CRFs,
all correspondences, dates of any monitoring visits, and records that support
this
CA 02335649 2001-O1-19
WO 00/04194 PCTNS99/16440
146
information for a period of 15 years following notification by the study
sponsor that
the clinical investigations have been completed or discontinued. All local
laws
regarding retention of records must also be followed.
X1 VL 13.4 Reporting and Communication of Results
All information concerning the study sponsor's perations, such as patent
applications, formulas, manufacturing processes, basic scientific data, and
formulation information supplied by the study sponsor and not published
previously,
are considered confidential and shall remain the sole property of the study
sponsor.
The investigator agrees to use this information only in conducting this study
and
shall not use it for any other purposes without the study sponsor's written
approval.
The investigator agrees not to disclose the study sponsor's confidential
information
to anyone except to people involved in the study who need such information to
assist
15 in conducting the study and then only on like terms of confidentiality and
nonuse.
It is understood by the investigator that the information developed from this
clinical
study will be used by the study sponsor and therefore may be disclosed as
required
to other clinical investigators, to the U.S. Food and Drug Administration, the
2o Canadian Health and Welfare Health Protection Branch, the European
Medicines
Evaluation Agency, and to other government agencies. In order to allow for the
use
of the information derived from the clinical studies, it is understood that
there is an
obligation to provide the study sponsor with complete test results and all
data
developed in the study.
No publication or disclosure of study results will be permitted except as
specified in
a separate, written agreement between the study sponsor and the investigator.
XLVII. 13.5 PROTOCOL COMPLETION
The IRB/REB/EC must be notified of completion or termination of the protocol.
Within 3 months of protocol completion or termination, the investigator must
provide a final clinical summary report to the IRB/REB/EC. The Principal
Investigator must maintain an accurate and complete record of all submissions
made
to the IRB/REB/EC, including a list of all reports and documents submitted. A
copy
of these reports should be sent to the study sponsor.
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
147
XLYI11. REFERENCES
Ausubel, F., et al. ( 1997) Current Protocols in Molecular Biology. Wiley and
Sons,
New York.
BritishMoscow, J.A., Connolly, T., Myers, T.G., et al. ( 1997) Reduced folate
carrier
gene (RFC 1 ) expression and anti-folate resistance in transfected and non-
slected cell
lines. Int. J. Cancer 72: 184-190.
Buroker et al., (1994) Journal of Clinical Oncology 12:14-20.
Campbell, L, Jones, T. Foulkes, W. and J. Trowsdale (1991) Folate binding
protein
is a marker for ovarian cancer. Cancer Reearch S 1: 5329-38.
Chang, F.-M. and Kidd, K.K. (1997) American Journal ofMedical Genetics 74:91-
94.
Diasio RB, Beavers TL, Carpenter JT.(1988) Familial deficiency of
dihydropyrimidine dehydrogenase. Biochemical basis for familial pyrimidinemia
and severe S-fluorouracil-induced toxicity. J Clin Invest 81:47-S 1.
Etienne, M.C., LaGrange, J.L., Dassonville, O., et al. (1994) Population study
of
dihydropyrimidine dehydrogenase in cancer patients. J. Clin. Oncology 12: 2248-
2253.
Falconer,D.S. and T.F.C. Mackay (1997) Introduction to Quantitative Genetics.
Longman, Essex.
Felipe, A., Valdes, R., Santo, B., et al. (1998) Na+ dependent nucleoside
transport in
liver: two different isoforms from the same gene family are expressed in liver
cells.
Biochem. J. 330: 997-1001.
HARRIS BE, CARPENTER JT, DIASIO RB. (1991) SEVERE S-
FLOUROURACIL TOXICITY SECONDARY TO DIHYDROPYRIMIDINE
DEHYDROGENASE DEFICIENCY. A POTENTIAL MORE COMMON
PHARMACOGENETIC SYNDROME. CANCER 68:499-SO1.
Haviland, M.B., Kessling, A.M., Davignon, J. and Sing, C. F. 1995. Cladistic
analysis of the apolipoprotein AI-CIII-AIV gene cluster using a healthy French
3o Canadian sample. I. Haploid analysis. Ann. Hum. Genet. 59: 211-231.
Keightley, P.D. (1996) Metabolic models of selection response. J. Theoretical
Biology 182: 311-316.
Kohne, C.H., Hiddemann, W., Schuller, J., et al. (1995) Failure of orally
administered dipyridamole to enhance the antineoplastic activity of
fluorouracil in
combination with leucovorin in patients with advanced colorectal cancer: a
prospective reandomized trial. J. Clin. Oncol. 13: 1201-1208.
CA 02335649 2001-O1-19
WO 00!04194 PCT/US99/16440
148 . -
Krynetski, E.Y., Tai, H.-L., Yates, C.R., et al. (1996) Genetic polymorphism
of
thiopurine S-methyltransferase: clinical importance and molecular mechanisms.
Pharmacogenetics 6: 279-290.
Lu, Z., Shang, R. and R.B. Diasio. (1993) Dihydropyrimidine dehydrogenase
activity in human peripheral blood mononuclear cells and liver: population
characteristics, newly identified deficient patients and clinical implications
The
genetic basis of Quantitative variation. TIG 11: 464-470.
Michalatos-Beloin, S. Tishkoff,S.A., Bentley, et al. (1996) Nucleic Acids
Research
24: 4841-4843
to Milano, G. and M.C. Etienne. (1994) Potential importance of
dihydropyrimidine
dehydrogenase (DPD) in cancer chemotherappy. Pharmacogenetics 4: 301-306.
Ridge, S.A., Brown, O., McMurrough, Fernandez-Salguero, P., Evans, W.E.,
Gonzalez, F.J. and H.L. McLeod ( 1997) Mutations at codon 974 of the DPYD gene
are a rare event. British Journal of Cancer 75: 178-179.
Ridge, S.A., Sludden, J., Wei, X., Sapone, A., Brown, O., Hardy, S., Canney,
P.,
Fernandez-Salguero, P., Gonzalez, F.J., Cassidy, J. and H.L. McLeod (1997)
Dihydropyrimidine dehydrogenase pharmacogenetics in patients with colorectal
cancer. British Journal of Cancer 77: 497-500.
Templeton, A. R. , Boerwinkle, E. and Sing, C.F. 1987. A cladistic analysis of
2o phenotypic associations with haplotypes inferred from restriction
endonuclease
mapping. I. Basic theory and an analysis of Alcohol Dehydrogenase activity in
Drosophila. Genetics 117: 343-351.
Terwilliger J., Ott J {1994) Handbook of Human Linkage Analysis. Baltimore:
John
Hopkins University Press.
Vreken P., Van Kuilenburg, A.B., Meinsma, R. and A.H. van Gennip (1997)
Dihydropyrimidine dehydrogenase (DPD) deficiency: identification and
expression
of missense mutations C29R, R886H and R235W. Human Genetics 101: 333-338.
Wang, J., Schaner, M.E., Thomassen, S., et al. (1997) Functional and molecular
characteristics of Na+ dependent nucleoside transporters. Pharmaceutical
Research
14: 1524-32.
Wei, X., McLeod, H.L., McMurrough, J., et al. (1996) Molecular basis of the
human
dihydropyrimidine dehydrogenase deficiency and 5-fluorouracil toxicity. J.
Clin.
Invest. 98: 610-615.
Wolmark, et al. (1996) Proceedings Am. Soc. Clin Oncol. 1 S: 460.
Yokata, H., Fernandez-Salguero, P., Furuya, H., Lin, K., McBride, O.M.,
Podschum,
B., Schnackerz, K.D., and Gonzalez, F.J. 1994. JBC 269:23192-23196
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
149
XLIX. SIGNED AGREEMENT OF THE STUDYPROTOCOL
I have read the foregoing protocol, VRG-9801, "Case-control study
to determine the relationship between toxicity of 5-fluorouracil (5-Ftn given
with folinic acid (FA) to patients with solid tumors and DNA sequence
variances in enzymes that mediate the action of 5-FU and FA", Version 1,
and agree~to conduct the study as detailed herein and to inform all who assist
me in the conduct of this study of their responsibilities and obligations.
to
t5
Principal Investigator's Signature Date
2o Principal Investigator's Name (Print)
Investigational Site (Print)
CA 02335649 2001-O1-19
WO 00/04194 _ PCT/US99/16440
150
APPENDIX II
This document describes procedures for handling blood samples from cancer
patients enrolled in trial for genetic studies at the study sponsor. The
approach will
be to first establish permanent lymphoblastoid cell lines. DNA and RNA will
subsequently be extracted from these cell lines. This procedure will save the
effort
of purifying DNA and RNA directly from blood. Since the pharmacogenetic
1o hypotheses to be investigated relate to the effect of genotype, not mRNA
expression
levels, lymphoblastoid cell lines should be satisfactory sources of nucleic
acid for
the genetic studies.
1. Cell line establishment will be done by the study site institutions (e.g.,
Genomics Core Facility of the Massachusetts General Hospital (MGH) Molecular
Neurogenetics Unit).
2. From each patient collect two 8.5 ml yellow topped tubes (containing ACD
solution A) for lymphoblastoid cell line development. All DNA and RNA will be
2o produced from the cell lines at a later date; therefore there is no need
for additional
blood drawing.
3. Fill out a DNA/Cell Line Order Sheet. An example is attached. Please note
that the patient's name should be anonymized at this point. (The Genomics Core
Facility will accept anonymized order forms.) All samples (including those for
PK
studies) should be assigned the same arbitrary number to allow subsequent
matching
of clinical, pharmacokinetic and genetic data. Also, the date and time of
blood
drawing should be marker. DOB should be recorded as month and year only, and
sex should be recorded. Record the number of tubes of blood drawn (2), date of
3o draw and date of shipment. Under "Requisition" check off "Transformation
only" .
4. Arrange for the two ACD blood samples to be delivered to designated
individual at the study site institution at the address given below:
Name and address of designated individual at study site institution.
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
151
Since the blood samples are typically aged at room temperature for a day or
two
before cell line establishment, it is not urgent that blood be delivered the
same day it
is drawn. Storage overnight, if necessary, should be at room temperature.
5. Please fax to the study sponsor a copy of the cell line order form so we
are
aware of accumulating cell lines. The fax number is 588-5399. Please fax to
the
attention of the designated individual jor the study sponsor.
6. Once cell lines are established, vials will be archived at the study site
l0 institution, where they will be available to investigators.
7. Questions for the study sponsor should be addressed to the designated
individual.
Example 11 Example 10
Hardy-Weinberg equilibrium
Evolution is the process of change and diversification of organisms through
time, and evolutionary change affects morphology, physiology and reproduction
of
organisms, including humans. These evolutionary changes are the result of
changes
in the underlying genetic or hereditary material. Evolutionary changes in a
group of
interbreeding individuals or Mendelian population, or simply populations, are
described in terms of changes in the frequency of genotypes and their
constituent
alleles. Genotype frequencies for any given generation is the result of the
mating
among members (genotypes) of their previous generation. Thus, the expected
proportion of genotypes from a random union of individuals in a given
population is
essential for describing the total genetic variation for a population of any
species.
For example, the expected number of genotypes that could form from the random
union of two alleles, A and a, of a gene are AA, Aa and aa. The expected
frequency
of genotypes in a large, random mating population was discovered to remain
constant from generation to generation; or achieve Hardy-Weinberg equilibrium,
named after its discoverers. The expected genotypic frequencies of alleles A
and a
(AA, 2Aa, aa) are conventionally described in terms of p~ + 2pq + q'- in which
p and
q are the allele frequencies of A and a. In this equation (p2 + 2pq + qz = 1
), p is
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99IIb440
152
defined as the frequency of one allele and q as the frequency of another
allele for a
trait controlled by a pair of alleles (A and a). In other words, p equals all
of the
alleles in individuals who are homozygous dominant (AA) and half of the
alleles in
individuals who are heterozygous (Aa) for this trait. In mathematical terms,
this is
p=AA+'/ZAa
Likewise, q equals the other half of the alleles for the trait in the
population, or
q = as + %Aa
Because there are only two alleles in this case, the frequency of one plus the
frequency of the other must equal 100%, which is to say
l0 p+q=1
Alternatively,
p=1-q OR q=1-p
All possible combinations of two alleles can be expressed as:
Is (P+q)~= 1
or more simply,
p=+2pq+qz= 1
In this equation, if p is assumed to be dominant, then pz is the frequency of
homozygous dominant (AA) individuals in a population, 2pq is the frequency of
2o heterozygous (Aa) individuals, and qZ is the frequency of homozygous
recessive (aa)
individuals.
From observations of phenotypes, it is usually only possible to know the
frequency of homozygous dominant or recessive individuals, because both
dominant
and recessives will express the distinguishable traits. However, the Hardy-
Weinberg
25 equation allows us to determine the expected frequencies of all the
genotypes, if
only p or q is known. Knowing p and q, it is a simple matter to plug these
values
into the Hardy-Weinberg equation (p= + 2pq + qz = 1 ). This then provides the
frequencies of all three genotypes for the selected trait within the
population.
This illustration shows Hardy-Weinberg frequency distributions for the
30 genotypes AA, Aa, and as at all values for frequencies of the alleles, p
and q. It
should be noted that the proportion of heterozygotes increases as the values
of p and
q approach 0.5.
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
153
Linkage disequilibirum
Linkage is the tendency of genes or DNA sequences (e.g. SNPs) to be
inherited together as a consequence of their physical proximity on a single
s chromosome. The closer together the markers are, the lower the probability
that
they will be separated during DNA crossing over, and hence the greater the
probability that they will be inherited together. Suppose a mutational event
introduces a "new" allele in the close proximity of a gene or an allele. The
new allele
will tend to be inherited together with the alleles present on the
"ancestral,"
to chromosome or haplotype. However, the resulting association, called linkage
disequilibrium, will decline over time due to recombination. Linkage
disequilibrium
has been used to map disease genes. In general, both allele and haplotype
frequencies differ among populations. Linkage disequilibrium is varied among
the
populations, being absent in some and highly significant in others.5
is
Quantification of the relative risk of observable outcomes of a
Pharmacogenetics
Trial
Let PiaR be the piacebo response rate (0% ( PIaR ( 100%) and TntR be the
treatment response rate (0% ( TntR ( 100%) of a classical clinical trial.
ObsRR is
2o defined as the relative risk between TntR and PIaR:
ObsRR = TntR / PIaR.
Suppose that in the treatment group there is a polymorphism in relation to
drug metabolism such as the treatment response rate is different for each
genotypic
subgroup of patients. Let q be the allele a frequency of a recessive biallelic
locus
2s {e.g. SNP) and p = 1 - q the allele A frequency. Following Hardy-Weinberg
equilibrium, the relative frequency of homozygous and heterozygous patients
are as
follow:
AA: p2 Aa: 2pq aa: q2
with
30 (p2+2pq+q2) = 1.
Let's define AAR, AaR, aaR as respectively the response rates of the AA, Aa
and as
patients. We have the following relationship:
CA 02335649 2001-O1-19
WO 00/04194 PCTNS99/16440
154
TntR = AAR*p2 + AaR*2pq + aaR*q2.
Suppose that the as genotypic group of patients has the lowest response rate,
i.e. a
response rate equal to the placebo response rate (which means that the
polymorphism has no impact on natural disease evolution but only on drug
action)
and let's define ExpRR as the relative risk between AAR and aaR, as
ExpRR = AAR / aaR.
From the previous equations, we have the following relationships:
ObsRR ( ExpRR ( 1/PIaR
TntR / PIaR = (AAR*p2 + AaR*2pq + aaR*q2) / PIaR
to The maximum of the expected relative risk, max(ExpRR), corresponding to the
case
of heterozygous patients having the same response rate as the placebo rate, is
such
that:
ObsRR = ExpRR*p2 + 2pq + q2 p ExpRR = (ObsRR - 2pq --q2) / p2
min(ExpRR),
corresponding to the case of heterozygous patients having the same response
rate as
the homozygous non-affected patients, is such that:
ObsRR = ExpRR*(p2 + 2pq) +q2 t~ ExpRR = (ObsRR -q2) / (p2 + 2pq)
For example, if q = 0.4, PIaR = 40% and ObsRR = 1.5 (i.e. TntR = 60%), then I
.6
2o ExpRR ( 2.4. This means that the best treatment response rate we can expect
in a
genotypic subgroup of patients in these conditions would be 95.6% instead of
60%.
This can also be expressed in terms of maximum potential gain between the
observed difference in response rates (TntR - PIaR) without any
phanmacogenetic
hypothesis and the maximum expected difference in response rates
(max(ExpRR)*PIaR - TntR) with a strong pharmacogenetic hypothesis:
(max(ExpRR)*PIaR - TntR) _ [(ObsRR - 2pq -q2) / p2] * PIaR - TntR
r~ (max(ExpRR)*PIaR - TntR) _ (TntR - PIaR*(2pq + q2) -TntR*p2]/p2
r~ (max(ExpRR)*PIaR - TntR) _ [TntR*( 1- p2}- PIaR*(2pq + q2)]/p2
t~ (max(ExpRR)*PIaR - TntR) _ [( I - p2) / p2] * (TntR - PIaR)
3o that is for the previous example, (95.6% - 60%) _ [(1 - 0.62)/0.62]* (60% -
40%) =
35.6%
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
155
Suppose that, instead of one SNP, we have p loci of SNPs for one gene. This
means that we have 2p possible haplotypes for this gene and (2p)(2p-1)/2
possible
genotypes. And with 2 genes with p 1 and p2 SNP loci, we have [(2p I )(2p 1-
1)/2]*[(2p2)(2p2-1)/2] possibilities; and so on. Examining haplotypes instead
of
combinations of SNPs is especially useful when there is linkage disequilibrium
enough to reduce the number of combinations to test, but not complete since in
this
latest case one SNP would be sufficient. Yet the problem of frequency above
still
remains with haplotypes instead of SNPs since the frequency of a haplotype
cannot
be higher than the highest SNP frequency involved.
to
Statistical Methods to be used in Objective Analyses
The statistical significance of the differences between
variance frequencies can be assessed by a Pearson chi-squared test of
homogeneity of proportions with n-1 degrees of freedom. Then, in order to
determine whip variances) is(are) responsible for an eventual significance,
we can consider each variance individually against the rest, up to n
comparisons, each based on a 2x2 table. This should result in chi-sequared
tests that are individually valid, but taking the most significant of these
tests
is a form of multiple testing. A Bonferroni's adjustment for multiple testing
will thus be made to the P-values, such as p*=1-(1-p)".
The statistical significance of the difference between genotype
frequencies associated to every variance can be assessed by a Pearson chi-
squared test of homogeneity of proportions with 2 degrees of freedom, using
the same Bonferroni's adjustment as above.
Testing for unequal haplotype frequencies between cases and
controls can be considered in the same framework as testing for unequal
variance frequencies since a single variance can be considered as a haplotype
of a single locus. The relevant likelihood ratio test compares a model where
two seqarate sets of haplotype frequencies apply to the cases and controls, to
one where the entire sample is characterized by a single common set of
haplotype frequencies. This can be performed by repeated use of a computer
program (Terwilliger and Ott, 1994, Handbook of Human Linkage Analysis,
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
156
Baltimore, John Hopkins University Press) to successively obtain the log-
likelihood corresponding to the set of haplotpe frequency estimates on the
cases (lnL~,~), on the controls (lnL~o""~,), and on the overall (lnL~~;"~).
The
test statistic 2((lnL~,~~)+ (lnL~~"~,)- (lnL~o,~b;"~)) is then chi-squared
with r-I
degrees of freedom (where r is the number of haplotypes).
To test for potentially confounding effects or effect-modifiers,
such as sex, age, etc., logistic regression can be used with case-control
status
as the outcome variable, and genotypes and covariates (plus possible
interactions) as predictor variables.
to
Example 12 Exemplary P6armacogenetic Analysis Steps
In accordance with the discussion of distribution frequencies
for variances, alleles, and haplotypes, variance detection, and correlation of
variances or haplotypes with treatment response variability, the points below
list major items which will typically be performed in an analysis of the
pharmacogenetic determination of the effects of variances in the treatment of
a disease and the selection/optimization of treatment.
~ List candidate gene/genes for a known genetic disease, and assign them to
the
respective metabolic pathways.
~ Determine their alleles, observed and expected frequencies, and their
relative
distributions among various ethnic groups, gender, both in the control and in
the
study (case) groups
~ Measure the relevant clinical/phenotypic (biochemical / physiological)
variables
of the disease
~ If the causal variance/allele in the candidate gene is unknown, then
determine
linkage disequilibria among variances of the candidate genes)
~ Divide the regions of the candidate genes into regions of high linkage
3o disequilibrium and low disequilibrium
~ Develop haplotypes among variances that show strong linkage disequilibrium
using the computation methods.
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
157
~ Determine the presence of rare haplotypes experimentally. Confirm if the
computationally determined rare haplotypes agree with the experimentally
determined haplotypes. If there is a disagreement between the experimentally
determined haplotypes and the computationally derived haplotypes, drop the
computationally derived rare haplotypes,
~ Construct cladograms from these haplotypes using the Templeton (1987)
algorithm.
~ Note regions of high recombination. Divide regions of high recombination
further to see patterns of linkage disequilibria.
to ~ Establish association between cladograms and clinical variables using the
nested
analysis of variance as presented by Templeton (1995), and assign causal
variance to a specific haplotype
~ For variances in the regions of high recombination, use permutation tests
for
establishing associations between variances and the phenotypic variables
~ If two or more genes are found to affect a clinical variable determine the
relative
contribution of each of the genes or variances in relation to the clinical
variable,
using step-wise regression or discriminant function or principal component
analysis.
~ Determine the relative magnitudes of the effects of any of the two variances
on
the clinical variable due to their genetic (additive, dominant or epistasis)
interaction.
~ Using the frequency of an allele or haplotypes, as well as
biochemical/clinical
variables determined in the in vitro or in vivo studies, determine the effect
of that
gene or allele on the expression of the clinical variable, according to the
measured genotype approach of Boerwinkle et al (Ann. Hum. Genet 1986).
~ Stratify ethnic/ clinical populations based on the presence or absence of a
given
allele or a haplotype
~ Optimize drug dosages based on the frequency of alleles and haplotypes as
well
as their effects using the measured genotype approach as a guide
Example 13 Method for Producing cDNA
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
158 - _
In order to identify sequence variances in a gene by laboratory methods it is
in some instances useful to produce cDNA(s) from multiple human subjects. (In
other instances it may be preferable to study genomic DNA.). Methods for
producing cDNA are known to those skilled in the art, as are methods for
amplifying
and sequencing the cDNA or portions thereof. An example of a useful cDNA
production protocol is provided below. As recognized by those skilled in the
art,
other specific protocols can also be used.
cDNA Production
** Make sure that all tubes and pipette tips are RNase-free. (Bake them
overnight at 100°C in a vaccum oven to make them RNase-free.)
1 Add the following to a RNase-free 0.2 ml micro-amp tube and mix gently:
24 ul water (DEPC treated)
12 ul RNA ( 1 ug/ul)
12 ul random hexamers(50 ng/ul)
2 Heat the mixture to 70°C for ten minutes.
3 Incubate on ice for 1 minute.
4 Add the following:
16 ul 5 X Synthesis Buffer
8 ul 0.1 M DTT
4 ul 10 mM dNTP mix ( 10 mM each dNTP)
4 ul SuperScript RT II enzyme
Pipette gently to mix.
5 Incubate at 42°C for 50 minutes.
6 Heat to 70°C for ten minutes to kill the enzyme, then place it on
ice.
7 Add 160 ul of water to the reaction so that the final volume is 240 ul.
8 Use PCR to check the quality of the cDNA. Use primer pairs that will give a
800 base pair long piece. See "PCR Optimization" for the PCR protocol.
The following chart shows the reagent amounts for a 20 ul reaction, a 80 ul
reaction, and a batch of 39 (which makes enough mix for 36) reactions:
20 ul X 1 tube 80 ul X I tube ~ 80u1 X 39 tubes
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99I16440 -
159
water 6 ul ~~ 24 ul ' 936 water
~~
RNA 3 ul 12 ul RNA
random hexamers3 ul ! 2 ul 468 random hexamers
synthesis 4 ul 16 u1 624 synthesis
buffer buffer
O.1MDTT 2u1 8u1 312 O.1MDTT
lp~ ~'p 1 ul 4 ul 156 IOmM dNTP
SSRT 1 ul 4 ul 156 SSRT
Example 14
Method for Detecting Variances by Single Strand Conformation
Polymorphism (SSCP) Analysis
This example describes the SSCP technique for identification of sequence
variances of genes. SSCP is usually paired with a DNA sequencing method, since
the SSCP method does not provide the nucleotide identity of variances. One
useful
sequencing method, for example, is DNA cycle sequencing of 'ZP labeled PCR
products using the Femtomole DNA cycle sequencing kit from Promega (W)7 and
the instructions provided with the kit. Fragments are selected for DNA
sequencing
based on their behavior in the SSCP assay.
Single strand conformation polymorphism screening is a widely used
technique for identifying an discriminating DNA fragments which differ from
each
other by as little as a single nucleotide. As originally developed by Orita et
al.
(Detection of polymorphisms of human DNA by gel electrophoresis as single-
strand
conformation polymorphisms. Proc Natl Acad Sci U S A. 86(8):2766-70, 1989),
the
technique was used on genomic DNA, however the same group showed that the
technique works very well on PCR amplified DNA as well. In the last 10 years
the
technique has been used in hundreds of published papers, and modifications of
the
technique have been described in dozens of papers. The enduring popularity of
the
technique is due to (1) a high degree of sensitivity to single base
differences (>90%)
(2) a high degree of selectivity, measured as a low frequency of false
positives, and
(3) technical ease. SSCP is almost always used together with DNA sequencing
because SSCP does not directly provide the sequence basis of differential
fragment
mobility. The basic steps of the SSCP procdure are described below.
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/1b440
160
When the intent of SSCP screening is to identify a large number of gene
variances it is useful to screen a relatively large number of individuals of
different
racial, ethnic and/or geographic origins. For example, 32 or 48 or 96
individuals is a
convenient number to screen because gel electrophoresis apparatus are
available with
96 wells (Applied Biosystems Division of Perkin Elmer Corporation), allowing 3
X
32, 2 X 48 or 96 samples to be loaded per gel.
The 32 (or more) individuals screened should be representative of most of the
worlds major populations. For example, an equal distribution of Africans,
Europeans and Asians constitutes a reasonable screening set. One useful source
of
l0 cell lines from different populations is the Coriell Cell Repository
(Camden, Nn,
which sells EBV immortalized lyphoblastoid cells obtained from several
thousand
subjects, and includes the raciaUethnic/geographic background of cell line
donors in
its catalog. Alternatively, a panel of cDNAs can be isolated frorii any
specific target
population.
IS SSCP can be used to analyze cDNAs or genomic DNAs. For many genes
cDNA analysis is preferable because for many genes the full genomic sequence
of
the target gene is not available, however, this circumstance will change over
the next
few years. To produce cDNA requires RNA. Therefore each cell lines is grown to
mass culture and RNA is isolated using an acid/phenol protocol, sold in kit
form as
2o Trizoi by Life Technologies (Gaithersberg, MD). The unfractionated RNA is
used to
produce cDNA by the action of a modified Maloney Murine Leukemia Virus
Reverse Transcriptase, purchased in kit form from Life Technologies
(Superscript II
kit). The reverse transcriptase is primed with random hexamer primers to
initiate
cDNA synthesis along the whole length of the RNAs. This proved useful later in
25 obtaining good PCR products from the 5' ends of some genes. Alternatively,
oligodT can be used to prime cDNA synthesis.
Material for SSCP analysis can be prepared by PCR amplification of the
cDNA in the presence of one a'~P labeled dNTP (usually a'ZP dCTP). Usually the
concentration of nonradioactive dCTP is dropped from 200 uM (the standard
3o concentration for each of the four dNTPs) to about 100 uM, and'~P dCTP is
added to
a concentration of about 0.1-0.3 uM. This involves adding a 0.3- 1 ul (3-10
uCi) of
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
161 - _
'2P cCTP to a 10 ul PCR reaction. Radioactive nucleotides can be purchased
from
DuPont/New England Nuclear.
The customary practice is to amplify about 200 base pair PCR products for
SSCP, however, an alternative approach is to amplify about 0.8-1.4 kb
fragments and
then use several cocktails of restriction endonucleases to digest those into
smaller
fragments of about 0.1-0.4kb, aiming to have as many fragments as possible
between
.15 and .3 kb. The digestion strategy has the advantage that less PCR is
required,
reducing both time and costs. Also, several different restriction enzyme
digests can
be performed on each set of samples (for example 96 cDNAs), and then each of
the
to digests can be run separately on SSCP gels. This redundant method (where
each
nucleotide is surveyed in three different fragments) reduces both the false
negative
and false positive rates. For example: a site of variance might lie within 2
bases of
the end of a fragment in one digest, and as a result not affect the
conformation of that
strand; the same variance, in a second or third digest, would likely lie in a
location
t5 more prone to affect strand folding, and therefore be detected by SSCP.
After digestion, the radiolabelled PCR products are diluted 1:5 by adding
fonnamide load buffer (80% formamide, 1X SSCP gel buffer) and then denatured
by
heating to 90%C for 10 minutes, and then allowed to renature by quickly
chilling on
ice. This procedure (both the dilution and the quick chilling) promotes infra-
(rather
2o than inter-) strand association and secondary structure formation. The
secondary
structure of the single strands influences their mobility on nondenaturing
gels,
presumably by influencing the number of collisions between the molecule and
the
gel matrix (i.e., gel sieving). Even single base differences consistently
produce
changes in intrastrand folding sufficient to register as mobility differences
on SSCP.
25 The single strands were then resolved on two gels, one a 5.5% acrylamide,
0.5X TBE gel, the other an 8% acrylamide, 10% glycerol, 1X TTE gel. (Other gel
recipes are known to those skilled in the art.) The use of two gels provides a
greater
opportunity to recognize mobility differences. Both glycerol and acrylamide
concentration have been shown to influence SSCP performance. By routinely
3o analyzing three different digests under two gel conditions (effectively 6
conditions),
and by looking at both strands under all 6 conditions, one can achieve a 12-
fold
CA 02335649 2001-O1-19
WO 00/04194 PCTlUS99/16440
162
sampling of each base pair of cDNA. However, if the goal is to rapidly survey
many
genes or cDNAs then a less redundant procedure would be optimal.
Example 15
Method for Detecting Variances by T4 endoauclease VII (T4E7) mismatch
cleavage method
The enzyme T4 endonuclease VII is derived from the bacteriophage T4. T4
endonuclease VII is used by the bacteriophage , to cleave branched DNA
intermediates which form during replication so the DNA can be processed and
l0 packaged. T4 endonuclease can also recognize and cleave heteroduplex DNA
containing single base mismatches as well as deletions and insertions. This
activity
of the T4 endonuclease VII enzyme can be exploited to detect sequence
variances
present in the general population.
The following are the major steps involved in identifying sequence variations
in a
candidate gene by T4 endonuclease VII mismatch cleavage:
1. Amplification by the polymerase chain reaction (PCR) of 400-600 by regions
of the candidate gene from a panel of DNA samples The DNA samples can
zo either be cDNA or genomic DNA and will represent some cross section of
the world population.
2. Mixing of a fluorescently labeled probe DNA with the sample DNA.
Heating and cooling the mixtures causing heteroduplex formation between
the probe DNA and the sample DNA.
3. Addition of T4 endonuclease VII to the heteroduplex DNA samples. T4
endonuclease will recognize and cleave at sequence variance mismatches
formed in the heteroduplex DNA.
4. Electrophoresis of the cleaved fragments on an ABI sequences to determine
the site of cleavage.
S. Sequencing of a subset of PCR fragments identified by T4 endonuclease VI
to contain variances to establish the specific base variation at that
location.
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
163
A more detailed description of the procedure is as follows:
A candidate gene sequence is downloaded from an appropriate database.
Primers for PCR amplification are designed which will result in the target
sequence
being divided into amplification products of between 400 and 600 bp. There
will be
a minimum of a 50 by of overlap not including the primer sequences between the
5'
and 3' ends of adjacent fragments to ensure the detection of variances which
are
located close to one of the primers.
Optimal PCR conditions for each of the primer pairs is determined
experimentally. Parameters including but not limited to annealing temperature,
pH,
1o MgCl2 concentration, and KCl concentration will be varied until conditions
for
optimal PCR amplification are established. The PCR conditions derived for each
primer pair is then used to amplify a panel of DNA samples {cDNA or genomic
DNA) which is chosen to best represent the various ethnic backgrounds of the
world
population or some designated subset of that population.
One of the DNA samples is chosen to be used as a probe. The same PCR
conditions used to amplify the panel are used to amplify the probe DNA.
However,
a flourescently labeled nucleotide is included in the deoxy-nucleotide mix so
that a
percentage of the incorporated nucleotides will be fluorescently labeled.
The labeled probe is mixed with the corresponding PCR products from each
of the DNA samples and then heated and cooled rapidly. This allows the
formation
of heteroduplexes between the probe and the PCR fragments from each of the DNA
samples. T4 endonuclease VII is added directly to these reactions and allowed
to
incubate for 30 min. at 37 C. 10 ul of the Fonmamide loading buffer is added
directly to each of the samples and then denatured by heating and cooling. A
portion
of each of these samples is electrophoresed on an ABI 377 sequences. If there
is a
sequence variance between the probe DNA and the sample DNA a mismatch will be
present in the heteroduplex fragment formed. The enzyme T4 endonuclease VII
will
recognize the mismatch and cleave at the site of the mismatch. This will
result in
the appearance of two peaks corresponding to the two cleavage products when
run
on the ABI 377 sequences.
Fragments identified as containing sequencing variances are
subsequently sequenced using conventional methods to establish the exact
CA 02335649 2001-O1-19
WO 00/04194 PCTNS99/1b440
1~ _
location and sequence variance.
Example 16
Method for Detecting Variances by DNA sequencing.
Sequencing by the Singer dideoxy method or the Maxim Gilbert chemical
cleavage method is widely used to determine the nucleotide sequence of genes.
Presently, a worldwide effort is being put forward to sequence the entire
human
genome. The Human Genome Project as it is called has already resulted in the
identification and sequencing of many new human genes. Sequencing can not only
be used to identify new genes, but can also be used to identify variations
between
individuals in the sequence of those genes.
The following are the major steps involved in identifying sequence variations
in a candidate gene by sequencing:
1. Amplification by the polymerise chain reaction (PCR) of 400-700 by regions
of the candidate gene from a panel of DNA samples The DNA samples can
either be cDNA or genomic DNA and will represent some cross section of
the world population.
2. Sequencing of the resulting PCR fragments using the Singer dideoxy
method. Sequencing reactions are performed using flourescently labeled
dideoxy terminators and electrophoresedon an ABI 377 sequences or its
equivalent.
3. Analysis of the resulting data from the ABI 377 sequences using software
programs designed to identify sequence variations between the different
samples analyzed.
A more detailed description of the procedure is as follows:
A candidate gene sequence is downloaded from an appropriate database.
Primers for PCR amplification are designed which will result in the target
sequence
being divided into amplification products of between 400 and 700 bp. There
will be
a minimum of a 50 by of overlap not including the primer sequences between the
5'
CA 02335649 2001-O1-19
WO 00/04!94 PCT/US99/16440
165
and 3' ends of adjacent fragments to ensure the detection of variances which
are
located close to one of the primers.
Optimal PCR conditions for each of the primer pairs is determined
experimentally. Parameters including but not limited to annealing temperature,
pH,
MgCh concentration, and KCl concentration will be varied until conditions for
optimal PCR amplification are established. The PCR conditions derived for each
primer pair is then used to amplify a panel of DNA samples (cDNA or genomic
DNA) which is chosen to best represent the various ethnic backgrounds of the
world
population or some designated subset of that population.
to PCR reactions are purified using the QIAquick 8 PCR purification kit
(Qiagen cat# 28142) to remove nucleotides, proteins and buffers. The PCR
reactions are mixed with 5 volumes of Buffer PB and applied to the wells of
the
QIAquick strips. The liquid is pulled through the strips by applying a vacuum.
The
wells are then washed two times with 1 ml of buffer PE and allowed to dry for
5
minutes under vacuum. The PCR products are eluted from the strips using 60 ul
of
elution buffer.
The purified PCR fragments are sequenced in both directions using the
Perkin Elmer ABI Prism'"'' Big Dye's'' terminator Cycle Sequencing Ready
Reaction
Kit (Cat# 4303150). The following sequencing reaction is set up: 8.0 ul
Terminator
2o Ready Reaction Mix, 6.0 ul of purified PCR fragment, 20 picomoles of
primer,
deionized water to 20 ul. The reactions are run through the following cycles
25
times: 96°C for 10 second, annealing temperature for that particular
PCR product for
5 seconds, 60°C for 4 minutes.
The above sequencing reactions are ethanol precipitated directly in the PCR
plate, washed with 70% ethanol, and brought up in a volume of 6 ul of
formamide
dye. The reactions are heated to 90°C for 2 minutes and then quickly
cooled to 4°C.
1 ul of each sequencing reaction is then loaded and run on an ABI 377
sequences.
The output for the ABI sequences appears as a series of peaks where each of
the different nucleotides, A, C, G, and T appear as a different color. The
nucleotide
3o at each position in the sequence is determined by the most prominent peak
at each
location. Comparison of each of the sequencing outputs for each sample can be
examined using software programs to determine the presence of a variance in
the
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
166 - -
sequence. One example of heterozygote detection using sequencing with dye
labeled terminators is described by Kwok et. al. (Kwok, P.-Y.; Carlson, C.;
Yager,
T.D., Ankener, W.,and D. A. Nickerson, Genomics 23, 138-144, 1994). The
software compares each of the normalized peaks between all the samples base by
base and looks for a 40% decrease in peak height and the concomitant
appearance of
a new peak underneath. Possible variances flagged by the software are further
analyzed visually to confirm their validity
In connection with the provision and description of nucleic acid sequences,
to the references herein to gene names and to GenBank and OMIM reference
numbers
provides the relevant sequences, recognizing that~the described sequences
will, in
most cases, also have other corresponding allelic variants. Also, it is
recognized that
the referenced sequences may contain sequencing error. Such error does not
interfere with identification of a relevant gene or portion of a gene, and can
be
readily corrected by redundant sequencing of the relevant sequence (preferably
using
both strands of DNA). Nucleic acid molecules or sequences can be readily
obtained
or determined utilizing the reference sequences. In general, molecules such as
nucleic acid hybridization probes and amplification primers can be provided
and are
described by the selected portion of the reference sequence, corrected if
necessary.
2o Thus, nucleic acid hybridization probes and/or primers are thus described
by a
portion of a reference sequence or a sequence complementary thereto (sequence
corrected if necessary), or an allelic variant of such a sequence, which
preferably
includes at least one variance site, preferably a variance site indicative of
the
effectiveness of a treatment for a disease or condition, and preferably
include at least
12,13,14,15,16,17,18,19,20,23,25,27,30,35,40,45, or SO nucleotides.
All patents and publications mentioned in the specification are indicative of
the levels of skill of those skilled in the art to which the invention
pertains. All
references cited in this disclosure are incorporated by reference to the same
extent as
if each reference had been incorporated by reference in its entirety
individually.
3o One skilled in the art would readily appreciate that the present invention
is
well adapted to carry out the objects and obtain the ends and advantages
mentioned,
as well as those inherent therein. The methods, variances, and compositions
CA 02335649 2001-O1-19 ,
WO 00/04194 PCT/US99/16440
167
described herein as presently representative of preferred embodiments are
exemplary
and are not intended as limitations on the scope of the invention. Changes
therein
and other uses will occur to those skilled in the art, which are encompassed
within
the spirit of the invention, are defined by the scope of the claims.
It will be readily apparent to one skilled in the art that varying
substitutions
and modifications may be made to the invention disclosed herein without
departing
from the scope and spirit of the invention. For example, using other
compounds,
and/or methods of administration are all within the scope of the present
invention.
Thus, such additional embodiments are within the scope of the present
invention and
to the following claims.
The invention illustratively described herein suitably may be practiced in the
absence of any element or elements, limitation or limitations which is not
specifically disclosed herein. Thus, for example, in each instance herein any
of the
terms "comprising", "consisting essentially of" and "consisting of may be
replaced with either of the other two terms. The terms and expressions which
have
been employed are used as terms of description and not of limitation, and
there is no
intention that in the use of such terms and expressions of excluding any
equivalents
of the features shown and described or portions thereof, but it is recognized
that
various modifications are possible within the scope of the invention claimed.
Thus,
it should be understood that although the present invention has been
specifically
disclosed by preferred embodiments and optional features, modification and
variation of the concepts herein disclosed may be resorted to by those skilled
in the
art, and that such modifications and variations are considered to be within
the scope
of this invention as defined by the appended claims.
In addition, where features or aspects of the invention are described in terms
of Markush groups or other grouping of alternatives, those skilled in the art
will
recognize that the invention is also thereby described in terms of any
individual
member or subgroup of members of the Markush group or other group.
Thus, additional embodiments are within the scope of the invention and
within the following claims.
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
168 -
Variance Table
Table 10
Name GID OMIM ID VGX Symbol Description
Variance Start Variance
CDS Context
D13811 D13811238310 GEN-AA Glycine cleavage system: Protein
T
277 1486>T V50L
D138I 1D13811238310 GEN-AA Glycine cleavage system: Protein
T
101073 9446>A R315K
D13811D13811238310 GEN-AA Glycine cleavage system: Protein
T
1083 9546>A S
D13811D13811238310 GEN-AA Glycine cleavage system: Protein
T
1773 1644C>T 3
15D13811D13811238310 GEN-AA Glycine cleavage system: Protein
T
2037 1908C>T 3
103626 103626 258900GEN-C6 Uridine monophosphate synthetase
(orotate phosphoribosyl
transferase and
orotidine-5-decarboxylase)
742 6386>C
G213A
20103626 103626 258900GEN-C6 Uridine monophosphate synthetase
(orotate phosphoribosyl
transferase and
orotidine-5-decarboxylase)
1575 1471A>G
3
103626 103626 258900GEN-C6 Uridine monophosphate synthetase
(orotate phosphoribosyl
transferase and
orotidine-5-decarboxylase)
1424 1320C>T
25S
104031 104031 None CB Methenyltetrahydrofolate cyclohydrolase
GEN-
454 4016>A R134K
104031 104031 None
GEN-CB Methenyltetrahydrofolate
cyclohydrolase
969 916C>G Q306E
30104031 104031 None
GEN-CB Methenyltetrahydrofolate
cyclohydrolase
1614 1561T>C S
104031 104031 None
GEN-CB Methenyltetrahydrofolate
cyclohydrolase
2011 19586>A R653Q
104031 104031 None
GEN-CB Methenyltetrahydrofolate
cyclohydrolase
352335 2282C>T T761M
K02581K02581188300 GEN-CI Thymidine kinase 1 ~ 90 33C>T
S
K02581K02581188300 GEN-CI Thymidine kinase 1 279 2226>A
S
K02581K02581188300 GEN-CI Thymidine kinase 1 282 2256>A
40S
K02581 K02581188300 GEN-CI Thymidine kinase 1 772 715A>G
3
K02581K02581188300 GEN-CI Thymidine kinase 1 867 8106>A
3
45K02581 K02581188300 GEN-CI Thymidine kinase 1 479 422C>T
P141L
K02581 K02581188300 GEN-CI Thymidine kinase 1 112 556>A
G 19R
K02581K02581188300 GEN-CI Thymidine kinase 1 487 4306>A
50E 144K
K02581 K02581188300 GEN-CI Thymidine kinase 1 445 388A>G
R130G
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
169
K02581K02581188300 GEN-CI Thymidine kinase 1 313 256C>T
F
K02581 K02581188300 GEN-CI Thymidine kinase 1 334 2776>T
V93F
K02581 K0258I 188300GEN-CI Thymidine kinase 1 329 272-
278TGGCTGT>TGGCTGT S
M64590 M64590 238300 GEN-FU Glycine cleavage system:
Protein P 3076 2926A>G
M976V
M69175 M69175 None GEN-FX Glycine cleavage system:
Protein
t H 710 686C>G 3
0
M69175 M69175 None GEN-FX Glycine cleavage system:
Protein
H 1007 983C>T 3
U09178U09178274270 GEN-HA Dihydropyrimidine Dehydrogenase
166 85'hC C29R
15U09178U09178274270 GEN-HA Dihydropyrimidine Dehydrogenase
577 496A>G M 166V
U09I78U09178274270 GEN-HA Dihydropyrimidine Dehydrogenase
3925 3844A>G 3
U09178U09178274270 GEN-HA Dihydropyrimidine Dehydrogenase
203937 3856'hC 3
U09178U09178274270 GEN-HA Dihydropyrimidine Dehydrogenase
1708 1627A>G I543V
U09178U09178274270 GEN-HA Dihydropyrimidine Dehydrogenase
3432 3351'1>C 3
25U09178U09178274270 GEN-HA Dihydropyrimidine Dehydrogenase
3730 36496>A 3
U09178U09178274270 GEN-HA Dihydropyrimidine Dehydrogenase
638 557A>G Y186C
U19720U19720600424 GEN-I1 Folate Transporter (SLC19A1)
341 246C>G
30S
U19720U19720600424 GEN-IIFolate Transporter (SLC19A1)
53 (-43)T>C
5
U 19720U I 9720600424GEN-I 1 Folate Transporter (SLC
19A 1 ) 175 806>A
R27H
35U 19720U 19720600424GEN-I 1 Folate Transporter (SLC
19A 1 ) 791 696C>T
S
U50929U50929None
GEN-JF Methionine
synthetase (aka
homocysteine
methyltransferase) 1991A>G 3
2017
U77088U77088None
GEN-K4 Thymidine
kinase 2 1480 1472T>C
3
40X02308X02308188350 GEN-KL Thymidylate synthetase 1066
961T>C
3
X02308X02308188350 GEN-KL Thymidylate synthetase 1136
1031A>G
3
X02308X02308188350 GEN-KL Thymidylate synthetase 1497
1392T'>A
453
X59543X59543None
GEN-M2 Ribonucleoside
diphosphate reductase
1037
850C>A S
X59543X59543None
GEN-M2 Ribonucleoside
diphosphate reductase
2410
22236>A S
50X59543X59543None
GEN-M2 Ribonucleoside
diphosphate reductase
2419
2232A>G S
X59543X59543None
GEN-M2 Ribonucleoside
diphosphate reductase
2717
2530T>A 3
CA 02335649 2001-O1-19
WO 00/04194 PCT/US99/16440
170
X59618X59618180390 GEN-M3 Ribonucleotide reductase M2 polypeptide
524 330C>G S
X59618X59618180390 GEN-M3 Ribonucleotide reductase M2 polypeptide
1636 1442C>T 3
X59618X59618180390 GEN-M3 Ribonucleotide reductase M2 polypeptide
2259 2065T>C 3
X59618X59618180390 GEN-M3 Ribonucleotide reductase M2 polypeptide
189 (-6)'hG 5
X90858X90858None GEN-NQ Uridine phosphorylase 1133 781'hA
C261S
X17620X17620None GEN-20M Human mRNA for Nm23 protein, involved in
developmental regulation (homolog. to Drosophila Awd protein)244 2446>T
D82Y
L38928 L38928 None GEN-2PT Homo sapiens 5,10-methenyltetrahydrofolate
synthetase mRNA, complete cds 617 604A>G T202A
572487 S72487 None GEN-3LD orfl 5 to PD-ECGF/'I'I'...orf15 to PD-ECGFfTP
[human, epidermoid carcinoma cell line A431, mRNA, 3 genes, 1718 nt]601 437G>C
3
M98045 M98045 None GEN-4C3 Homo Sapiens folylpoiyglutamate
synthetase mRNA, complete cds 1747 I677G>T 3
M98045 M98045 None GEN-4C3 Homo sapiens folylpolyglutamate
synthetase mRNA, complete cds 1900 1830T>C 3
L11931 L11931 None GEN-4DT Human cytosolic serine hydroxymethyltransferase
(SHMT) mRNA, complete cds 1444 1420C>T L474F
L11931 L11931 None GEN-4DT Human cytosolic serine hydroxymethyltransferase
(SHMT) mRNA, complete cds 1541 1517C>T 3
DHFR J00140 126060 GEN-4E9 Human dihydrofolate reductase gene
721 679'hA 3
DHFR J00140 126060 GEN-4E9 Human dihydrofolate reductase gene
829 787C>T 3
U09806U09806None GEN-4FZ Human methylenetetrahydrofolate reductase
mRNA, partial cds 1289 1289C>A E430A
U09806U09806None GEN-4FZ Human methylenetetrahydrofolate reductase
mRNA, partial cds 473 4736>A R158Q
U09806U09806None GEN-4FZ Human methylenetetrahydrofolate reductase
mRNA, partial cds 550 SSOC>T F
U09806U09806None GEN-4FZ Human methylenetetrahydrofolate reductase
mRNA, partial cds 668 668C>T A223V
U09806U09806None GEN-4FZ Human methylenetetrahydrofolate reductase
mRNA, partial cds 1308 1308'hC 3
U09806U09806None GEN-4FZ Human methylenetetrahydrofolate reductase
mRNA, partial cds 120 120'hC S
U09806U09806None GEN-4FZ Human methylenetetrahydrofolatc reductase
mRNA, partial cds 1059 1059'T>C S